Rapid Method for Detection of Pathogen

ABSTRACT

The present specification discloses methods of detecting a pathogen of interest and components useful in carrying out these methods, including a pre-enrichment media, and enrichment media and a detection solution.

This patent application claims priority to U.S. Ser. No. 14/161,117,filed Jan. 22, 2014, and pursuant to 35 U.S.C. §119(e) claims priorityto U.S. Provisional Patent Application 61/927,401, filed Jan. 14, 2014,and claims priority pursuant to 35 U.S.C. §119(a) to Spanish PatentApplication ES 201330064, filed Jan. 22, 2013, each of which is herebyincorporated by reference in its entirety.

Pathogens can contaminate food or food animals during production,processing and preparation. Similarly, pathogens can contaminate watersources or seafood harvested from such contaminated waters. Humanexposure to pathogens can cause illness, most often gastroenteritis, butalso potentially more serious diseases such as salmonellosis andhepatitis A. Exposure to pathogens can occur either by direct contactwith, or ingestion of, contaminated foods or water or indirectly basedon cross-contamination. Even though the United States has one of thesafest food supplies in the world, there are still millions of cases offoodborne illness each year.

Early and rapid detection of pathogen contamination is vitial to preventwidespread outbreaks of illness and ensure the health of the public atlarge. During the past 25 years, pathogen assays that increasesensitiviety and specificity as well as decrease the time involved toperform these assays have been developed by experts in appliedmicrobiology and microbiological analysis. Although there is nouniversally accepted definition for these so-called rapid assays, thesemethods are simply performed faster than the traditional ones, or areeasier to implement, or are more sensitive and specific. Despite thename, currently available rapid assays for pathogen detection stillrequire several days to complete the assay and determine whether a foodor water supply is contaminated with a pathogen.

The main obstacles associated with reducing the time needed to conductpathogen detection assays is the balance between the sensitivity,specificity, and lower limit of detection of the assay on one hand andhaving enough pathogen present to detect its presence abovecontaminating microorganisms and background noise.

The present specification discloses a rapid method for the detection ofpathogens which provides high sensitivity and specificity, a lower limitof detection, yet can be performed more quickly then currently availablemethods.

SUMMARY

Aspects of the present specification disclose a method of detecting apathogen in a sample. In aspects, a method disclosed herein comprisesthe steps of a) incubation of the sample in a pre-enrichment comprisinga low growth nutrient component, a growth inhibiting agent, and asurfactant with bacteriostatic or bactericidal action, wherein theincubation occurs at a specific temperature for a specific length oftime; b) incubating an aliquot of pre-enrichment media from step (a) inan enrichment media, the enrichment media comprising a high growthnutrient component and a growth promoting agent, wherein the incubationoccurs at a specific temperature for a specific length of time; and c)detecting the presence of absence of a pathogen by analyzing an aliquotof enrichment media from step (b).

Other aspects of the present specification disclose a pathogen analysiskit. In aspects, pathogen analysis kit comprising a pre-enrichment mediaand an enrichment media. In other aspects, the pathogen analysis kit mayfurther comprise detection solution. In yet other aspects, the pathogenanalysis kit may further comprise an immunopurification reagent system.In yet other aspects, the pathogen analysis kit may further comprise alabel or an insert providing instructions on how to use the kit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the proportionality between different densitiesof bacterial population and the signal obtained chronoamperometric.

FIG. 2 shows a graph of the proportionality between the registeredelectrochemical signal and progress in a particular bacterial loadproliferation.

FIG. 3 shows a graph of the linear time-response curve of the presenceof pathogen in fecal samples.

FIGS. 4A, 4B, and 4C show graphs of three independent experimentsplotting the presence of target DNA amplified during each cycle of a PCRreaction.

FIG. 5 shows a graph of the proportionality between the registeredelectrochemical signal and progress in a particular bacterial loadproliferation at pH 5.7. Bacterium were contacted with 1 mM pAPP, pH 5.7for 30 minutes at 37° C. CA E=0.2 V t=60 s PalmSens PS06333.

FIG. 6 shows a graph of the proportionality between the registeredelectrochemical signal and progress in a particular bacterial loadproliferation at pH 7.0. Bacterium were contacted with 1 mM pAPP, pH 5.7for 30 minutes at 37° C. CA E=0.2 V t=60 s PalmSens PS06333.

FIG. 7 shows a graph of the proportionality between the registeredelectrochemical signal and progress in a particular bacterial loadproliferation at pH 9.8. Bacterium were contacted with 1 mM pAPP, pH 5.7for 30 minutes at 37° C. CA E=0.2 V t=60 s PalmSens PS06333.

DESCRIPTION

The present specification discloses a method of detecting a pathogen ina sample. The method comprises a pre-enrichment step and an enrichmentstep that use selective pre-growth and growth media. The combination ofthese steps, together with specific time and temperature conditions,progressively increases the population of the pathogen as well as aneffective reduces the populations of unwanted organisms and/or otherbackground noise which interferes with the detection of the pathogen.The selective increase of the pathogen population as well as theeffectively removing unwanted organisms and/or other background noiseallows for a more sensitive and precise detection of the pathogen thencurrently available methods. In addition, the method disclosed hereinallows for a more rapid detection of a pathogen since it may becompleted in about eight hours to about 30 hours; current pathogendetection methods require about 2 days to about 5 days to complete.

Aspect of the present specification disclose, in part, a pathogen. Apathogen refers to a microorganism that can cause disease in its host.Non-limiting examples of a pathogen include a prion, a virus, abacterium, a fungus, a protazoan, a helimenth, and a parasite.

A prion is composed of a protein in a misfolded form. Prions reproduceby hijacking the functions of living cells and propagating a misfoldedprotein state by inducing existing, properly-folded proteins to convertinto the disease-associated, prion form. As such, the prion acts as atemplate to guide the misfolding of more proteins into prion form. Thesenewly formed prions can then go on to convert more proteins themselves,which triggers a chain reaction that produces large amounts of the prionform. Prions cause neurodegenerative disease by aggregatingextracellularly within the central nervous system to form plaques knownas amyloid, which disrupt the normal tissue structure. Prions areresponsible for the transmissible spongiform encephalopathies in avariety of mammals, including bovine spongiform encephalopathy (BSE,also known as “mad cow disease”) in cattle, scrapies in sheep and goats,and chronic wasting disease in deer. In humans, prions causeCreutzfeldt-Jakob disorders, Gerstmann-Sträussler-Scheinker syndrome,Fatal Familial Insomnia, and kuru.

A virus is a small infectious agent typically range between 20-300nanometers in length that replicates only inside the living cells ofother organisms. Virus particles (known as virions) consist of two orthree parts: i) the genetic material made from either DNA or RNA, longmolecules that carry genetic information; ii) a protein coat thatprotects these genes; and in some cases iii) an envelope of lipids thatsurrounds the protein coat when they are outside a cell. The shapes ofviruses range from simple helical and icosahedral forms to more complexstructures. The average virus is about one one-hundredth the size of theaverage bacterium. Viruses can infect all types of life forms, fromanimals and plants to bacteria and archaea. Non-limiting examples ofpathogenic viruses belong to the families Adenoviridae, Picornaviridae,Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae,Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus,Rhabdoviridae, and Togaviridae.

A bacteria is a single-celled prokaryotic microorganism characterized bythe lack of a membrane-bound nucleus and membrane-bound organelles andcan have a cell wall (Gram positive) or lack one (Gram negative).Morphologically, bacteria can be divided into rod-shaped (bacillus),round (coccus), spiral (spirillum), and incomplete spiral (vibrios).Non-limiting examples of pathogenic bacteria belong to the generaBacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia,Chlamydophila, Clostridium, Corynebacterium, Enterobacter, Enterococcus,Escherichia, Francisella, Haemophilus, Helicobacter, Legionella,Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Treponema, Vibrio, and Yersinia. Non-limiting examples of specificpathogenic bacterial species include a strain of Bacillus anthracis, astrain of a strain of Bordetella pertussis, a strain of a strain ofBorrelia burgdorferi, a strain of a strain of Brucella abortus, a strainof a strain of Brucella canis, a strain of a strain of Brucellamelitensis, a strain of a strain of Brucella suis, a strain of a strainof Campylobacter jejuni, a strain of Chlamydia pneumonia, a strain ofChlamydia trachomatis, a strain of Chlamydophila psittaci, a strain ofClostridium botulinum, a strain of Clostridium difficile, a strain ofClostridium perfringens, a strain of Clostridium tetani, a strain ofCorynebacterium diphtheria, a strain of Enterobacter sakazakii, a strainof Enterococcus faecalis, a strain of Enterococcus faecium, a strain ofEscherichia coli, a strain of Francisella tularensis, a strain ofHaemophilus influenza, a strain of Helicobacter pylori, a strain ofLegionella pneumophila, a strain of Leptospira interrogans, a strain ofListeria monocytogenes, a strain of Mycobacterium leprae, a strain ofMycobacterium tuberculosis, a strain of Mycobacterium ulcerans, a strainof Mycoplasma pneumonia, a strain of Neisseria gonorrhoeae, a strain ofNeisseria meningitides, a strain of Pseudomonas aeruginosa, a strain ofRickettsia rickettsia, a strain of Salmonella typhi and Salmonellatyphimurium, a strain of Shigella sonnei, a strain of Staphylococcusaureus, a strain of Staphylococcus epidermidis, a strain ofStaphylococcus saprophyticus, a strain of Streptococcus agalactiae, astrain of Streptococcus pneumonia, a strain of Streptococcus pyogenes, astrain of Treponema pallidum, a strain of Vibrio cholera, a strain ofYersinia enterocolitica, and, a strain of Yersinia pestis.

A fungus is a eukaryotic microorganism characterized by membrane-boundnucleus and organelles which lack chlorophyll have cell walls composedof chitin and reproduce by spores. Non-limiting examples of pathogenicfungi belong to the genera Asperfillus, Canidia, Cryptococcus,Histoplasma, Pneumocystis, and Stachybotrys. Non-limiting examples ofspecific pathogenic fungi species include a strain of Aspergillusclavatus, Aspergillus fumigatus, Aspergillus flavus, Canidia albicans,Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii,Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis jirovecii,Pneumocystis carinii, and Stachybotrys chartarum.

A protozoa is a eukaryotic single-cell microorganism characterized bymembrane-bound nucleus and organelles which lack chlorophyll and a cellwall and are motile. Protozoa commonly range from 10 to 52 micrometers,but can grow as large as 1 mm. Non-limiting examples of pathogenicprotozoa belong to the genera Acanthamoeba, Balamuthia, Cryptosporidium,Dientamoeba, Endolimax, Entamoeba, Giardia, Iodamoeba, Leishmania,Naegleria, Plasmodium, Sappinia, Toxoplasma, Trichomonas, andTrypanosoma. Non-limiting examples of specific pathogenic protozoaspecies include a strain of Acanthamoeba spp., Balamuthia mandrillaris,Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium hominis,Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidiumparvum, Dientamoeba fragilis, Endolimax nana, Entamoeba dispar,Entamoeba hartmanni, Entamoeba histolytica, Entamoeba coli, Entamoebamoshkovskii, Giardia lamblia, Iodamoeba butschlii, Leishmaniaaethiopica, Leishmania braziliensis, Leishmania chagasi, Leishmaniadonovani, Leishmania infantum, Leishmania major, Leishmania mexicana,Leishmania tropica, Naegleria fowleri, Plasmodium falciparum, Plasmodiumknowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax,Sappinia diploidea, Toxoplasma gondii, Trichomonas vaginalis,Trypanosoma brucei, and Trypanosoma cruzi.

Aspect of the present specification disclose, in part, a sample. Avariety of samples are useful in the methods disclosed herein. A samplerefers to a biological matter that contains or potentially contains apathogen. A sample encompasses but is not limited to a purifiedpathogen, a partially purified pathogen, a cell, a crude cell lysate, apartially purified cell lysate, a crude culture media, a partiallypurified culture media, a raw foodstuff, a partially-cooked foodstuff, acooked foodstuff, a processed foodstuff; a dairy foodstuff, a beverage,an animal feed, a fecal sample, a vegetative sample, a soil sample, awater sample, a pond sediment, a human tissue sample, a raw livestocktissue sample, a processed livestock tissue sample, such as, e.g.,leather.

Aspect of the present specification disclose, in part, a pre-enrichmentstep. A pre-enrichment step comprises incubating a sample disclosedherein in a pre-enrichment media for a defined time and at a definedtemperature. A pre-enrichment media, also referred to as apre-enrichment culture media is a buffered culture media that providesthe nutrients necessary to sustain low-growth of the pathogen. Inaddition, a pre-enrichment media may also contain components whichreduce or inhibit the growth of contaminating bacteria or othermicroorganisms. A pre-enrichment media comprises a low growth nutrientcomponent, a surfactant, and optionally a growth inhibiting agent and/ora growth enhancing agent.

A pre-enrichment media typically comprises a low growth nutrientcomponent used as a source of proteins, amino acids and nitrogen. Eithera single low growth nutrient component may comprise a pre-enrichmentmedia disclosed herein, or a plurality of low growth nutrient componentsmay comprise a pre-enrichment media disclosed herein.

A non-limiting example of a low growth nutrient component is a peptone,such as, e.g., a peptide from an animal source and a peptone from aplant source. A peptone from an animal source includes, withoutlimitation, an acid casein peptone, a bacteriological peptone, a beefextract powder, a casein peptone, a casein cc peptone, a gelatinpeptone, a meat peptone, a polypeptone proteose peptone, and a proteosepeptone No 3. A peptone from a plant source includes, withoutlimitation, a malt extract, a soya peptone, and a yeast extract.

Any concentration of low growth nutrient component may be used, with theproviso that the concentration is useful to practice the methodsdisclosed herein. In aspects of this embodiment, a low growth nutrientcomponent may be used at a concentration of, e.g., about 1 g/L, about 2g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L,about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L,about 13 g/L, about 14 g/L, or about 15 g/L. In other aspects of thisembodiment, a low growth nutrient component may be used at aconcentration of, e.g., at least 1 g/L, at least 2 g/L, at least 3 g/L,at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12g/L, at least 13 g/L, at least 14 g/L, or at least 15 g/L. In yet otheraspects of this embodiment, a low growth nutrient component may be usedat a concentration of, e.g., at most 1 g/L, at most 2 g/L, at most 3g/L, at most 4 g/L, at most 5 g/L, at most 6 g/L, at most 7 g/L, at most8 g/L, at most 9 g/L, at most 10 g/L, at most 11 g/L, at most 12 g/L, atmost 13 g/L, at most 14 g/L, or at most 15 g/L.

In yet other aspects of this embodiment, a low growth nutrient componentmay be used at a concentration of between, e.g., about 1 g/L to 2 g/L,about 1 g/L to 3 g/L, about 1 g/L to 4 g/L, about 1 g/L to 5 g/L, about1 g/L to 6 g/L, about 1 g/L to 7 g/L, about 1 g/L to 8 g/L, about 1 g/Lto 9 g/L, about 1 g/L to 10 g/L, about 1 g/L to 11 g/L, about 1 g/L to12 g/L, about 1 g/L to 13 g/L, about 1 g/L to 14 g/L, about 1 g/L to 15g/L, about 2 g/L to 3 g/L, about 2 g/L to 4 g/L, about 2 g/L to 5 g/L,about 2 g/L to 6 g/L, about 2 g/L to 7 g/L, about 2 g/L to 8 g/L, about2 g/L to 9 g/L, about 2 g/L to 10 g/L, about 2 g/L to 11 g/L, about 2g/L to 12 g/L, about 2 g/L to 13 g/L, about 2 g/L to 14 g/L, about 2 g/Lto 15 g/L, about 3 g/L to 4 g/L, about 3 g/L to 5 g/L, about 3 g/L to 6g/L, about 3 g/L to 7 g/L, about 3 g/L to 8 g/L, about 3 g/L to 9 g/L,about 3 g/L to 10 g/L, about 3 g/L to 11 g/L, about 3 g/L to 12 g/L,about 3 g/L to 13 g/L, about 3 g/L to 14 g/L, about 3 g/L to 15 g/L,about 4 g/L to 5 g/L, about 4 g/L to 6 g/L, about 4 g/L to 7 g/L, about4 g/L to 8 g/L, about 4 g/L to 9 g/L, about 4 g/L to 10 g/L, about 4 g/Lto 11 g/L, about 4 g/L to 12 g/L, about 4 g/L to 13 g/L, about 4 g/L to14 g/L, about 4 g/L to 15 g/L, about 5 g/L to 6 g/L, about 5 g/L to 7g/L, about 5 g/L to 8 g/L, about 5 g/L to 9 g/L, about 5 g/L to 10 g/L,about 5 g/L to 11 g/L, about 5 g/L to 12 g/L, about 5 g/L to 13 g/L,about 5 g/L to 14 g/L, about 5 g/L to 15 g/L, about 6 g/L to 7 g/L,about 6 g/L to 8 g/L, about 6 g/L to 9 g/L, about 6 g/L to 10 g/L, about6 g/L to 11 g/L, about 6 g/L to 12 g/L, about 6 g/L to 13 g/L, about 6g/L to 14 g/L, about 6 g/L to 15 g/L, about 7 g/L to 8 g/L, about 7 g/Lto 9 g/L, about 7 g/L to 10 g/L, about 7 g/L to 11 g/L, about 7 g/L to12 g/L, about 7 g/L to 13 g/L, about 7 g/L to 14 g/L, about 7 g/L to 15g/L, about 8 g/L to 9 g/L, about 8 g/L to 10 g/L, about 8 g/L to 11 g/L,about 8 g/L to 12 g/L, about 8 g/L to 13 g/L, about 8 g/L to 14 g/L,about 8 g/L to 15 g/L, about 9 g/L to 10 g/L, about 9 g/L to 11 g/L,about 9 g/L to 12 g/L, about 9 g/L to 13 g/L, about 9 g/L to 14 g/L,about 9 g/L to 15 g/L, about 10 g/L to 11 g/L, about 10 g/L to 12 g/L,about 10 g/L to 13 g/L, about 10 g/L to 14 g/L, about 10 g/L to 15 g/L,about 11 g/L to 12 g/L, about 11 g/L to 13 g/L, about 11 g/L to 14 g/L,about 11 g/L to 15 g/L, about 12 g/L to 13 g/L, about 12 g/L to 14 g/L,about 12 g/L to 15 g/L, about 13 g/L to 14 g/L, about 13 g/L to 15 g/L,or about 14 g/L to 15 g/L.

A pre-enrichment media may also comprise a surfactant. Surfactants arecompounds that lower the surface tension of a liquid, allowing easierspreading, and lowering of the interfacial tension between two liquids,or between a liquid and a solid. Either a single surfactant may comprisea pre-enrichment media disclosed herein, or a plurality of surfactantsmay comprise a pre-enrichment media disclosed herein. A surfactantdisclosed herein provides bacteriostatic and bactericide action thatretards or prevents growth of unwanted organisms contained in a sampledisclosed herein. As non-limiting examples, a surfactant may retard orprevent growth of unwanted bacterial cells by disrupting the mechanismsof action of adsorption on the surface, interfering with the osmoticbalance, preventing the intake of nutrients, denaturing proteins,inhibiting enzyme activity, and/or damaging the cell membrane.

Useful surfactants, include, without limitation, ionic surfactants,zwitterionic (amphoteric) surfactants, non-ionic surfactants, or anycombination therein. The surfactant used in a method disclosed hereincan be varied as appropriate by one skilled in the art and generallydepends, in part, on the particular pre-enrichment media being used, thepathogen being detected, and/or the particular unwanted bacteria beingremoved. Ionic surfactants include anionic surfactants based onpermanent (sulfate, sulfonate, phosphate) or pH dependent (carboxylate)anions. Anionic surfactants include, without limitation, alkyl sulfateslike ammonium lauryl sulfate and sodium lauryl sulfate (SDS); alkylether sulfates like sodium laureth sulfate and sodium myreth sulfate;docusates like dioctyl sodium sulfosuccinate; tetradecyl sodium dodecylsulfate; 7-ethyl-2-methyl-4-undecyl sodium sulphate; octadecyl sulphate;sulfonate fluorosurfactants like perfluorooctanesulfonate (PFOS) andperfluorobutanesulfonate; alkyl benzene sulfonates; alkyl aryl etherphosphates; alkyl ether phosphates; alkyl carboxylates like fatty acidsalts and sodium stearate; sodium lauroyl sarcosinate; and carboxylatefluorosurfactants like perfluorononanoate and perfluorooctanoate.

Ionic surfactants also include cationic surfactants based on permanentor pH dependent cations. Cationic surfactants include, withoutlimitation, alkyltrimethylammonium salts like cetyl trimethylammoniumbromide (CTAB) and cetyl trimethylammonium chloride (CTAC);cetylpyridinium chloride (CPC); polyethoxylated tallow amine (POEA);benzalkonium chloride (BAC); benzethonium chloride (BZT);5-Bromo-5-nitro-1,3-dioxane; dimethyldioctadecylammonium chloride; anddioctadecyldimethylammonium bromide (DODAB), as well as pH-dependentprimary, secondary or tertiary amines like surfactants where the primaryamines become positively charged at pH greater than 10, or the secondaryamines become charged at pH less than 4, like octenidinedihydrochloride.

Zwitterionic surfactants are based on primary, secondary or tertiaryamines or quaternary ammonium cation with a sulfonate, a carboxylate, ora phosphate. Zwitterionic surfactants include, without limitation,3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);sultaines like cocamidopropyl hydroxysultaine; betaines likecocamidopropyl betaine; or lecithins.

Non-ionic surfactants are less denaturing and as such are useful tosolubilize membrane proteins and lipids while retaining protein-proteininteractions. Non-limiting examples of surfactants includepolyoxyethylene glycol sorbitan alkyl esters like polysorbate 20sorbitan monooleate (TWEEN® 20), polysorbate 40 sorbitan monooleate(TWEEN® 40), polysorbate 60 sorbitan monooleate (TWEEN® 60), polysorbate61 sorbitan monooleate (TWEEN® 61), polysorbate 65 sorbitan monooleate(TWEEN® 65), polysorbate 80 sorbitan monooleate (TWEEN® 80), andpolysorbate 81 sorbitan monooleate (TWEEN® 81); poloxamers(polyethylene-polypropylene copolymers), like Poloxamer 124 (PLURONIC®L44), Poloxamer 181 (PLURONIC® L61), Poloxamer 182 (PLURONIC® L62),Poloxamer 184 (PLURONIC® L64), Poloxamer 188 (PLURONIC® F68), Poloxamer237 (PLURONIC® F87), Poloxamer 338 (PLURONIC® L108), and Poloxamer 407(PLURONIC® F127); alkyl phenol polyglycol ethers; polyethylene glycolalkyl aryl ethers; polyoxyethylene glycol alkyl ethers, likeoctaethylene glycol monododecyl ether, pentaethylene glycol monododecylether, BRIJ® 30, and BRIJ® 35; 2-dodecoxyethanol (LUBROL®-PX);polyoxyethylene glycol octylphenol ethers like polyoxyethylene (4-5)p-t-octyl phenol (TRITON® X-45) and polyoxyethylene octyl phenyl ether(TRITON® X-100); nonylphenol ethoxylates like Nonoxynol-4 (TERGITOL™NP-4), Nonoxynol-6 (TERGITOL™ NP-6), Nonoxynol-7 (TERGITOL™ NP-7),Nonoxynol-8 (TERGITOL™ NP-8), Nonoxynol-9 (TERGITOL™ NP-9), Nonoxynol-10(TERGITOL™ NP-10), Nonoxynol-11 (TERGITOL™ NP-11), Nonoxynol-12(TERGITOL™ NP-12), Nonoxynol-13 (TERGITOL™ NP-13), Nonoxynol-15(TERGITOL™ NP-15), Nonoxynol-30 (TERGITOL™ NP-30), Nonoxynol-40(TERGITOL™ NP-40), Nonoxynol-50 (TERGITOL™ NP-50), Nonoxynol-55(TERGITOL™ NP-55), and Nonoxynol-70 (TERGITOL™ NP-70);phenoxypolyethoxylethanols like nonyl phenoxypolyethoxylethanol andoctyl phenoxypolyethoxylethanol; glucoside alkyl ethers like octylglucopyranoside; maltoside alkyl ethers like dodecyl maltopyranoside;thioglucoside alkyl ethers like heptyl thioglucopyranoside; digitonins;glycerol alkyl esters like glyceryl laurate; alkyl aryl polyethersulfates; alcohol sulfonates; sorbitan alkyl esters; cocamideethanolamines like cocamide monoethanolamine and cocamidediethanolamine; sucrose monolaurate; dodecyl dimethylamine oxide, andsodium cholate. Other non-limiting examples of surfactants useful in themethods disclosed herein can be found in, e.g., Winslow, et al., Methodsand Compositions for Simultaneously Isolating Hemoglobin from Red BloodCells and Inactivating Viruses, U.S. 2008/0138790; Pharmaceutical DosageForms and Drug Delivery Systems (Howard C. Ansel et al., eds.,Lippincott Williams & Wilkins Publishers, 7^(th) ed. 1999); Remington:The Science and Practice of Pharmacy (Alfonso R. Gennaro ed.,Lippincott, Williams & Wilkins, 20^(th) ed. 2000); Goodman & Gilman'sThe Pharmacological Basis of Therapeutics (Joel G. Hardman et al., eds.,McGraw-Hill Professional, 10th ed. 2001); and Handbook of PharmaceuticalExcipients (Raymond C. Rowe et al., APhA Publications, 4^(th) edition2003), each of which is hereby incorporated by reference in itsentirety.

Any concentration of surfactant may be used, with the proviso that theconcentration is useful to practice the methods disclosed herein. Inaspects of this embodiment, a surfactant may be used at a concentrationof, e.g., about 0.01% (v/v), about 0.05% (v/v), about 0.075% (v/v),about 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v),about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v),about 0.9% (v/v), about 1.0% (v/v), about 2.0% (v/v), about 3.0% (v/v),about 4.0% (v/v), about 5.0% (v/v), about 6.0% (v/v), about 7.0% (v/v),about 8.0% (v/v), about 9.0% (v/v), or about 10.0% (v/v). In otheraspects of this embodiment, a surfactant may be used at a concentrationof, e.g., at least 0.01% (v/v), at least 0.05% (v/v), at least 0.075%(v/v), at least 0.1% (v/v), at least 0.25% (v/v), at least 0.5% (v/v),at least 0.75% (v/v), at least 1.0% (v/v), at least 2.5% (v/v), at least5.0% (v/v), at least 7.5% (v/v), or at least 10.0% (v/v). In yet otheraspects of this embodiment, a surfactant may be used at a concentrationof, e.g., at most 0.01% (v/v), at most 0.05% (v/v), at most 0.075%(v/v), at most 0.1% (v/v), at most 0.25% (v/v), at most 0.5% (v/v), atmost 0.75% (v/v), at most 1.0% (v/v), at most 2.5% (v/v), at most 5.0%(v/v), at most 7.5% (v/v), or at most 10.0% (v/v).

In still other aspects of this embodiment, a surfactant may be used at aconcentration of between, e.g., about 0.01% (v/v) to about 0.05% (v/v),about 0.01% (v/v) to about 0.1% (v/v), about 0.01% (v/v) to about 0.5%(v/v), about 0.01% (v/v) to about 1.0% (v/v), about 0.01% (v/v) to about2.0% (v/v), about 0.01% (v/v) to about 3.0% (v/v), about 0.01% (v/v) toabout 4.0% (v/v), about 0.01% (v/v) to about 5.0% (v/v), about 0.05%(v/v) to about 0.1% (v/v), about 0.05% (v/v) to about 0.5% (v/v), about0.05% (v/v) to about 1.0% (v/v), about 0.05% (v/v) to about 2.0% (v/v),about 0.05% (v/v) to about 3.0% (v/v), about 0.05% (v/v) to about 4.0%(v/v), about 0.05% (v/v) to about 5.0% (v/v), about 0.1% (v/v) to about0.5% (v/v), about 0.1% (v/v) to about 1.0% (v/v), about 0.1% (v/v) toabout 2.0% (v/v), about 0.2% (v/v) to about 0.5% (v/v), about 0.2% (v/v)to about 1.0% (v/v), about 0.2% (v/v) to about 2.0% (v/v), about 0.2%(v/v) to about 3.0% (v/v), about 0.2% (v/v) to about 4.0% (v/v), about0.2% (v/v) to about 5.0% (v/v), about 0.5% (v/v) to about 1.0% (v/v),about 0.5% (v/v) to about 2.0% (v/v), about 0.5% (v/v) to about 3.0%(v/v), about 0.5% (v/v) to about 4.0% (v/v), about 0.5% (v/v) to about5.0% (v/v), about 0.5% (v/v) to about 6.0% (v/v), about 0.5% (v/v) toabout 7.0% (v/v), about 0.5% (v/v) to about 8.0% (v/v), about 0.5% (v/v)to about 9.0% (v/v), about 0.5% (v/v) to about 10.0% (v/v), about 1.0%(v/v) to about 2.0% (v/v), about 1.0% (v/v) to about 3.0% (v/v), about1.0% (v/v) to about 4.0% (v/v), about 1.0% (v/v) to about 5.0% (v/v),about 1.0% (v/v) to about 6.0% (v/v), about 1.0% (v/v) to about 7.0%(v/v), about 1.0% (v/v) to about 8.0% (v/v), about 1.0% (v/v) to about9.0% (v/v), or about 1.0% (v/v) to about 10.0% (v/v).

In aspects of this embodiment, the surfactant may be used at aconcentration of, e.g., about 0.001 g/L, about 0.002 g/L, about 0.003g/L, about 0.004 g/L, about 0.005 g/L, about 0.006 g/L, about 0.007 g/L,about 0.008 g/L, about 0.009 g/L, about 0.01 g/L, about 0.02 g/L, about0.03 g/L, about 0.04 g/L, about 0.05 g/L, about 0.06 g/L, about 0.07g/L, about 0.08 g/L, about 0.09 g/L, or about 0.1 g/L. In other aspectsof this embodiment, the surfactant may be used at a concentration of,e.g., at least 0.001 g/L, at least 0.002 g/L, at least 0.003 g/L, atleast 0.004 g/L, at least 0.005 g/L, at least 0.006 g/L, at least 0.007g/L, at least 0.008 g/L, at least 0.009 g/L, at least 0.01 g/L, at least0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, atleast 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L,or at least 0.1 g/L. In yet other aspects of this embodiment, thesurfactant may be used at a concentration of, e.g., at most 0.001 g/L,at most 0.002 g/L, at most 0.003 g/L, at most 0.004 g/L, at most 0.005g/L, at most 0.006 g/L, at most 0.007 g/L, at most 0.008 g/L, at most0.009 g/L, at most 0.01 g/L, at most 0.02 g/L, at most 0.03 g/L, at most0.04 g/L, at most 0.05 g/L, at most 0.06 g/L, at most 0.07 g/L, at most0.08 g/L, at most 0.09 g/L, or at most 0.1 g/L.

In still other aspects of this embodiment, the surfactant may be used ata concentration of, e.g., about 0.001 g/L to about 0.005 g/L, about0.001 g/L to about 0.006 g/L, about 0.001 g/L to about 0.007 g/L, about0.001 g/L to about 0.008 g/L, about 0.001 g/L to about 0.009 g/L, about0.001 g/L to about 0.01 g/L, about 0.001 g/L to about 0.02 g/L, about0.001 g/L to about 0.03 g/L, about 0.001 g/L to about 0.04 g/L, about0.001 g/L to about 0.05 g/L, about 0.001 g/L to about 0.06 g/L, about0.001 g/L to about 0.07 g/L, about 0.001 g/L to about 0.08 g/L, about0.001 g/L to about 0.09 g/L, about 0.001 g/L to about 0.1 g/L, about0.005 g/L to about 0.01 g/L, about 0.005 g/L to about 0.02 g/L, about0.005 g/L to about 0.03 g/L, about 0.005 g/L to about 0.04 g/L, about0.005 g/L to about 0.05 g/L, about 0.005 g/L to about 0.06 g/L, about0.005 g/L to about 0.07 g/L, about 0.005 g/L to about 0.08 g/L, about0.005 g/L to about 0.09 g/L, about 0.005 g/L to about 0.1 g/L, about0.01 g/L to about 0.05 g/L, about 0.01 g/L to about 0.06 g/L, about 0.01g/L to about 0.07 g/L, about 0.01 g/L to about 0.08 g/L, about 0.01 g/Lto about 0.09 g/L, about 0.01 g/L to about 0.1 g/L, or about 0.05 g/L toabout 0.1 g/L.

A pre-enrichment media may optionally comprises a growth inhibitingagent. A growth inhibiting agent typically comprises a component thatreduces or inhibits the growth of contaminating bacteria or othercontaminating microorganisms. In addition, a growth inhibiting agent maynot affect the growth of a pathogen of interest or affects it to alesser extent that the contaminating bacteria or other contaminatingmicroorganisms. Either a single growth inhibiting agent may comprise apre-enrichment media disclosed herein, or a plurality of growthinhibiting agents may comprise a pre-enrichment media disclosed herein.Non-limiting examples of a growth inhibiting agent include ananti-microbial compound, iodine, magneusium, and a triarylmethane dye.

An anti-microbial compound is one that is antagonistic to the growth ofa microorganism. These compounds can be divided into two broadcategories of microbicidal agents which kill a microorganism andmicrobiostatic agents which slow down or stall the growth of amicroorganism. An anti-microbial compound is commonly classified basedon its mechanism of action, chemical structure, or spectrum of activity.Most target bacterial functions or growth processes. Those that targetthe bacterial cell wall (penicillins and cephalosporins) or the cellmembrane (polymyxins), or interfere with essential bacterial enzymes(rifamycins, lipiarmycins, quinolones, and sulfonamides) havebactericidal activities. Those that target protein synthesis(macrolides, lincosamides and tetracyclines) are usually bacteriostatic(with the exception of bactericidal aminoglycosides). Furthercategorization is based on their target specificity. “Narrow-spectrum”antibacterial antibiotics target specific types of bacteria, such asGram-negative or Gram-positive bacteria, whereas broad-spectrumantibiotics affect a wide range of bacteria. An anti-microbial compoundincludes, without limitation, an aminoglycoside, an ansamycin, acarbacephem, a carbapenen, a cephalosporin, a cyclic lipopeptide, aglycopeptide, a glycylcycline, a lincosamide, a lipiamycin, alipopeptide, a macrolide, a monobactam, a nitrofuran, an oxazolidonome,a penicillin, a qunolones, a sulfonamide, and a tetracycline.

Iodine combines with amino acids like tyrosine or histidine by a simplechemical reaction that denatures proteins having these amino acidsexposed to the extra-cellular environment.

Magnesium known to have anti-bacterial activity. In aspects of thisembodiment, magnesium is magnesium chloride and magnesium sulfate.

A triarylmethane dye is a group of synthetic organic compoundscontaining triphenylmethane backbones that produce an intense,pH-dependent color. Triarylmethane dyes can be grouped into familiesaccording to the nature of the substituents on the aryl groups. Methylviolet dyes have dimethylamino groups at the p-positions of two arylgroups and include, without limitation, methyl violet 2B, methyl violet6B, and methyl violet 10B. Fuchsine dyes have amine (NH2 or NHMe)functional groups at the p-positions of each aryl group and include,without limitation, pararosaniline, fuchsine, new fuchsine, fuchsinbasic violet, and fuchine acid. Phenol dyes have hydroxyl groups at thep positions of each aryl group and include, without limitation, phenolred, chlorophenol red, and cresol red. Malachite green dyes are relatedto the methyl violet dyes, except that they contain one phenyl (C₆H₅)group and include, without limitation, malachite green and brilliantgreen. A triarylmethane dye includes, without limitation, aluminon,aniline Blue WS, aurin, aurintricarboxylic acid, brilliant blue FCF,brilliant green, bromocresol green, bromocresol purple, bromophenolblue, bromopyrogallol red, bromothymol blue, bromsulphthalein,chlorophenol red, coomassie brilliant blue, cresol red, crystal violet,crystal violet lactone, ethyl green, fast green FCF, fluoran, fuchsine,fuchsine acid, gentian, green S, light green SF yellowish, malachitegreen, methyl blue, methyl violet, new fuchsine, pararosaniline, patentblue V, phenol red, phenolphthalein, rose bengal, thymolphthalein,victoria blue BO, water blue, xylene cyanol, and xylenol orange.

Any concentration of growth inhibiting agent may be used, with theproviso that the concentration is useful to practice the methodsdisclosed herein. In aspects of this embodiment, a growth inhibitingagent may be used at a concentration of, e.g., about 0.01% (v/v), about0.05% (v/v), about 0.075% (v/v), about 0.1% (v/v), about 0.2% (v/v),about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v),about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1.0% (v/v),about 2.0% (v/v), about 3.0% (v/v), about 4.0% (v/v), about 5.0% (v/v),about 6.0% (v/v), about 7.0% (v/v), about 8.0% (v/v), about 9.0% (v/v),or about 10.0% (v/v). In other aspects of this embodiment, a growthinhibiting agent may be used at a concentration of, e.g., at least 0.01%(v/v), at least 0.05% (v/v), at least 0.075% (v/v), at least 0.1% (v/v),at least 0.25% (v/v), at least 0.5% (v/v), at least 0.75% (v/v), atleast 1.0% (v/v), at least 2.5% (v/v), at least 5.0% (v/v), at least7.5% (v/v), or at least 10.0% (v/v). In yet other aspects of thisembodiment, a growth inhibiting agent may be used at a concentration of,e.g., at most 0.01% (v/v), at most 0.05% (v/v), at most 0.075% (v/v), atmost 0.1% (v/v), at most 0.25% (v/v), at most 0.5% (v/v), at most 0.75%(v/v), at most 1.0% (v/v), at most 2.5% (v/v), at most 5.0% (v/v), atmost 7.5% (v/v), or at most 10.0% (v/v).

In still other aspects of this embodiment, a growth inhibiting agent maybe used at a concentration of between, e.g., about 0.01% (v/v) to about0.05% (v/v), about 0.01% (v/v) to about 0.1% (v/v), about 0.01% (v/v) toabout 0.5% (v/v), about 0.01% (v/v) to about 1.0% (v/v), about 0.01%(v/v) to about 2.0% (v/v), about 0.01% (v/v) to about 3.0% (v/v), about0.01% (v/v) to about 4.0% (v/v), about 0.01% (v/v) to about 5.0% (v/v),about 0.05% (v/v) to about 0.1% (v/v), about 0.05% (v/v) to about 0.5%(v/v), about 0.05% (v/v) to about 1.0% (v/v), about 0.05% (v/v) to about2.0% (v/v), about 0.05% (v/v) to about 3.0% (v/v), about 0.05% (v/v) toabout 4.0% (v/v), about 0.05% (v/v) to about 5.0% (v/v), about 0.1%(v/v) to about 0.5% (v/v), about 0.1% (v/v) to about 1.0% (v/v), about0.1% (v/v) to about 2.0% (v/v), about 0.2% (v/v) to about 0.5% (v/v),about 0.2% (v/v) to about 1.0% (v/v), about 0.2% (v/v) to about 2.0%(v/v), about 0.2% (v/v) to about 3.0% (v/v), about 0.2% (v/v) to about4.0% (v/v), about 0.2% (v/v) to about 5.0% (v/v), about 0.5% (v/v) toabout 1.0% (v/v), about 0.5% (v/v) to about 2.0% (v/v), about 0.5% (v/v)to about 3.0% (v/v), about 0.5% (v/v) to about 4.0% (v/v), about 0.5%(v/v) to about 5.0% (v/v), about 0.5% (v/v) to about 6.0% (v/v), about0.5% (v/v) to about 7.0% (v/v), about 0.5% (v/v) to about 8.0% (v/v),about 0.5% (v/v) to about 9.0% (v/v), about 0.5% (v/v) to about 10.0%(v/v), about 1.0% (v/v) to about 2.0% (v/v), about 1.0% (v/v) to about3.0% (v/v), about 1.0% (v/v) to about 4.0% (v/v), about 1.0% (v/v) toabout 5.0% (v/v), about 1.0% (v/v) to about 6.0% (v/v), about 1.0% (v/v)to about 7.0% (v/v), about 1.0% (v/v) to about 8.0% (v/v), about 1.0%(v/v) to about 9.0% (v/v), or about 1.0% (v/v) to about 10.0% (v/v).

A pre-enrichment media may optionally comprises a growth enhancingagent. A growth enhancing agent promotes rapid growth of a pathogen byreducing the lag phase in culture media, thereby reactivating dormantpathogen.

Non-limiting examples of a growth enhancing agent include a siderophore.A siderophore is a high-affinity iron chelating compound that acts tosequester and solubilize the iron. These compounds are important to apathogen for its acquisition of iron in order to maintain cellularrespiration and DNA synthesis. This is because under most cultureenvironment, the amount of free iron (approximately 1×10⁻⁹ M) is belowthe concentration required by most bacterial pathogens for growth.Non-limiting examples of a siderophore include Aerobactin, Alcaligin,Azotobactin, Bacillibactin, Desferrioxamine B, Desferrioxamine E,Enterobactin, Ferrichrome, Ferrioxiamina-B, Ferrioxiamina-E, FusarinineC, Mycobactin, Ornibactin, Petrobactin, Pyoverdine, Pyochelin,Salmochelin, Staphyloferring A, Vibriobactin, and Yersiniabactin.

Any concentration of growth enhancing agent may be used, with theproviso that the concentration is useful to practice the methodsdisclosed herein. In aspects of this embodiment, a growth enhancingagent may be used at a concentration of, e.g., about 0.01 μM, about 0.05μM, about 0.075 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9μM, about 1.0 μM, about 2.0 μM, about 3.0 μM, about 4.0 μM, about 5.0μM, about 6.0 μM, about 7.0 μM, about 8.0 μM, about 9.0 μM, or about10.0% (v/v). In other aspects of this embodiment, a growth enhancingagent may be used at a concentration of, e.g., at least 0.01 μM, atleast 0.05 μM, at least 0.075 μM, at least 0.1 μM, at least 0.25 μM, atleast 0.5 μM, at least 0.75 μM, at least 1.0 μM, at least 2.5 μM, atleast 5.0 μM, at least 7.5 μM, or at least 10.0% (v/v). In yet otheraspects of this embodiment, a growth enhancing agent may be used at aconcentration of, e.g., at most 0.01 μM, at most 0.05 μM, at most 0.075μM, at most 0.1 μM, at most 0.25 μM, at most 0.5 μM, at most 0.75 μM, atmost 1.0 μM, at most 2.5 μM, at most 5.0 μM, at most 7.5 μM, or at most10.0 μM

In still other aspects of this embodiment, a growth enhancing agent maybe used at a concentration of between, e.g., about 0.01% (v/v) to about0.05 μM, about 0.01% (v/v) to about 0.1 μM, about 0.01% (v/v) to about0.5 μM, about 0.01% (v/v) to about 1.0 μM, about 0.01% (v/v) to about2.0 μM, about 0.01% (v/v) to about 3.0 μM, about 0.01% (v/v) to about4.0 μM, about 0.01% (v/v) to about 5.0 μM, about 0.05% (v/v) to about0.1 μM, about 0.05% (v/v) to about 0.5 μM, about 0.05% (v/v) to about1.0 μM, about 0.05% (v/v) to about 2.0 μM, about 0.05% (v/v) to about3.0 μM, about 0.05% (v/v) to about 4.0 μM, about 0.05% (v/v) to about5.0 μM, about 0.1% (v/v) to about 0.5 μM, about 0.1% (v/v) to about 1.0μM, about 0.1% (v/v) to about 2.0 μM, about 0.2% (v/v) to about 0.5 μM,about 0.2% (v/v) to about 1.0 μM, about 0.2% (v/v) to about 2.0 μM,about 0.2% (v/v) to about 3.0 μM, about 0.2% (v/v) to about 4.0 μM,about 0.2% (v/v) to about 5.0 μM, about 0.5% (v/v) to about 1.0 μM,about 0.5% (v/v) to about 2.0 μM, about 0.5% (v/v) to about 3.0 μM,about 0.5% (v/v) to about 4.0 μM, about 0.5% (v/v) to about 5.0 μM,about 0.5% (v/v) to about 6.0 μM, about 0.5% (v/v) to about 7.0 μM,about 0.5% (v/v) to about 8.0 μM, about 0.5% (v/v) to about 9.0 μM,about 0.5% (v/v) to about 10.0 μM, about 1.0% (v/v) to about 2.0 μM,about 1.0% (v/v) to about 3.0 μM, about 1.0% (v/v) to about 4.0 μM,about 1.0% (v/v) to about 5.0 μM, about 1.0% (v/v) to about 6.0 μM,about 1.0% (v/v) to about 7.0 μM, about 1.0% (v/v) to about 8.0 μM,about 1.0% (v/v) to about 9.0 μM, or about 1.0% (v/v) to about 10.0 μM).

In an embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of apeptone, 0.05 to 3% (v/v) of a growth inhibiting agent and 0.05 to 5%(v/v) of a surfactant. In aspects of this embodiment, a pre-enrichmentmedia comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to 3% (v/v) of ananti-microbial compound and 0.05 to 5% (v/v) of a surfactant. In otheraspects of this embodiment, a pre-enrichment media comprises 2 g/L to 12g/L of a soy peptone, 0.05 to 3% (v/v) of an anti-microbial compound and0.05 to 5% (v/v) of a non-ionic surfactant. In yet other aspects of thisembodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soypeptone, 0.05 to 3% (v/v) of an anti-microbial compound and 0.05 to 5%(v/v) of an ionic surfactant.

In aspects of this embodiment, a pre-enrichment media comprises 2 g/L to12 g/L of a soy peptone, 0.05 to 3% (v/v) of a triarylmethane dye and0.05 to 5% (v/v) of a non-ionic surfactant. In other aspects of thisembodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soypeptone, 0.05 to 3% (v/v) of triarylmethane dye and 0.05 to 5% (v/v) ofan ionic surfactant. In yet other aspects of this embodiment, apre-enrichment media comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to3% (v/v) of triarylmethane dye and 0.05 to 5% (v/v) of an nonylphenolethoxylate.

In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to8 g/L of a soy peptone, 0.05 to 3% (v/v) of a Malachite green dye and0.05 to 5% (v/v) of an nonylphenol ethoxylate. In other aspects of thisembodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soypeptone, 0.05 to 3% (v/v) of brilliant green and 0.05 to 5% (v/v) of annonylphenol ethoxylate. In aspects of this embodiment, a pre-enrichmentmedia comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of aMalachite green dye and 0.05 to 5% (v/v) of an octadecyl sulfate. Inother aspects of this embodiment, a pre-enrichment media comprises 4 g/Lto 8 g/L of a soy peptone, 0.05 to 3% (v/v) of brilliant green and 0.05to 5% (v/v) of an octadecyl sulfate.

In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to8 g/L of a soy peptone, 0.05 to 3% (v/v) of a Malachite green dye and0.05 to 5% (v/v) of a Nonoxynol-4 (Tergitol-4). In other aspects of thisembodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soypeptone, 0.05 to 3% (v/v) of Brilliant Green and 0.05 to 5% (v/v) of aNonoxynol-4 (Tergitol-4). In aspects of this embodiment, apre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to3% (v/v) of a Malachite green dye and 0.05 to 5% (v/v) of a tetradecylsodium dodecyl sulfate. In other aspects of this embodiment, apre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to3% (v/v) of Brilliant Green and 0.05 to 5% (v/v) of a tetradecyl sodiumdodecyl sulfate. In aspects of this embodiment, a pre-enrichment mediacomprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of aMalachite green dye and 0.05 to 5% (v/v) of a7-ethyl-2-methyl-4-undecyll sodium sulphate. In other aspects of thisembodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soypeptone, 0.05 to 3% (v/v) of Brilliant Green and 0.05 to 5% (v/v) of a7-ethyl-2-methyl-4-undecyll sodium sulphate.

In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of apeptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of a growthinhibiting agent and 0.05 mL/L to 6 mL/L of a surfactant. In aspects ofthis embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of aCaseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L ofan anti-microbial compound and 0.05 mL/L to 6 mL/L of a surfactant. Inother aspects of this embodiment, a pre-enrichment media comprises 5 g/Lto 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to0.0012 g/L of an anti-microbial compound and 0.05 mL/L to 6 mL/L of anon-ionic surfactant. In yet other aspects of this embodiment, apre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of an anti-microbialcompound and 0.05 mL/L to 6 mL/L of an ionic surfactant.

In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to0.0012 g/L of a triarylmethane dye and 0.05 mL/L to 6 mL/L of asurfactant. In other aspects of this embodiment, a pre-enrichment mediacomprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L BileSalts, 0.001 g/L to 0.0012 g/L of a triarylmethane dye and 0.05 mL/L to6 mL/L of a non-ionic surfactant. In yet other aspects of thisembodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of aCaseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of atriarylmethane dye and 0.05 mL/L to 6 mL/L of an ionic surfactant. Inaspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012g/L of a triarylmethane dye and 0.05 mL/L to 6 mL/L of a nonylphenolethoxylate.

In aspects of this embodiment, a pre-enrichment media comprises 8 g/L to12 g/L of a Caseine Peptone, 3 g/L to 5 g/L Bile Salts, 0.004 g/L to0.0008 g/L of a Malachite green dye and 1.0 mL/L to 3 mL/L of anonylphenol ethoxylate. In aspects of this embodiment, a pre-enrichmentmedia comprises 8 g/L to 12 g/L of a Caseine Peptone, 3 g/L to 5 g/LBile Salts, 0.004 g/L to 0.0008 g/L of Brilliant Green and 1.0 mL/L to 3mL/L of a nonylphenol ethoxylate.

In aspects of this embodiment, a pre-enrichment media comprises 8 g/L to12 g/L of a Caseine Peptone, 3 g/L to 5 g/L Bile Salts, 0.004 g/L to0.0008 g/L of a Malachite green dye and 1.0 mL/L to 3 mL/L ofNonoxynol-4 (Tergitol-4). In other aspects of this embodiment, apre-enrichment media comprises 8 g/L to 12 g/L of a Caseine Peptone, 3g/L to 5 g/L Bile Salts, 0.004 g/L to 0.0008 g/L of Brilliant Green and1.0 mL/L to 3 mL/L of Nonoxynol-4 (Tergitol-4).

In another aspect of this embodiment, a pre-enrichment media comprises10 g/L Caseine Peptone, 4 g/L Bile Salts, 3.5 g/L Disodium Phosphate,1.5 g/L Potassium Phosphate, 0.006 g/L Brilliant Green, and 2 mL/LNonoxynol-4 (Tergitol-4). In another aspect of this embodiment, apre-enrichment media comprises 10 g/L Caseine Peptone, 4 g/L Bile Salts,3.5 g/L Disodium Phosphate, 1.5 g/L Potassium Phosphate, 0.002 g/LBrilliant Green, and 2 mL/L Nonoxynol-4 (Tergitol-4).

In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of apeptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2g/L to 6 g/L of a first growth inhibiting agent, and 0.001 g/L to 0.0012g/L of a second growth inhibiting agent. In aspects of this embodiment,a pre-enrichment media comprises 5 g/L to 15 g/L of a Casien peptone, 2g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6g/L of an iodine compound, and 0.001 g/L to 0.0012 g/L of ananti-microbial compound. In other aspects of this embodiment, apre-enrichment media comprises 5 g/L to 15 g/L of a Casien peptone, 2g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6g/L of an iodine compound, and 0.001 g/L to 0.0012 g/L of atriarylmethane dye.

In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of apeptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2g/L to 6 g/L of a first growth inhibiting agent, 2 g/L to 6 g/L of asecond growth inhibiting agent, and 0.001 g/L to 0.0012 g/L of a thirdgrowth inhibiting agent. In aspects of this embodiment, a pre-enrichmentmedia comprises 5 g/L to 15 g/L of a Caseine peptone, 2 g/L to 6 g/L ofBile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a firstiodine compound, 2 g/L to 6 g/L of a second iodine compound, and 0.001g/L to 0.0012 g/L of an anti-microbial compound. In other aspects ofthis embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of aCaseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of MeatExtract, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of asecond iodine compound, and 0.001 g/L to 0.0012 g/L of a triarylmethanedye.

In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of apeptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2g/L to 6 g/L of a first growth inhibiting agent, 2 g/L to 6 g/L of asecond growth inhibiting agent, 0.001 g/L to 0.0012 g/L of a thirdgrowth inhibiting agent, and 0.001 g/L to 0.0012 g/L of a fourth growthinhibiting agent. In aspects of this embodiment, a pre-enrichment mediacomprises 5 g/L to 15 g/L of a Caseine peptone, 2 g/L to 6 g/L of BileSalts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first iodinecompound, 2 g/L to 6 g/L of a second iodine compound, 0.001 g/L to0.0012 g/L of an anti-microbial compound, and 0.001 g/L to 0.0012 g/L ofa triarylmethane dye.

In aspects of this embodiment, a pre-enrichment media comprises 7 g/L to11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5g/L Meat Extract, 3 g/L to 5 g/L of a first iodine compound, 3 g/L to 5g/L of a second iodine compound, 0.006 g/L to 0.0010 g/L of ananti-microbial compound, 0.006 g/L to 0.0010 g/L of a triarylmethanedye. In other aspects of this embodiment, a pre-enrichment mediacomprises 7 g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L BileSalts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5g/L of Potassium Iodide, 0.006 g/L to 0.0010 g/L of an aminocoumarinantibiotic, 0.006 g/L to 0.0010 g/L of a Malachite green dye. In yetother aspects of this embodiment, a pre-enrichment media comprises 7 g/Lto 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to5 g/L Meat Extract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L ofPotassium Iodide, 0.006 g/L to 0.0010 g/L of Novobiocin, 0.006 g/L to0.0010 g/L of Brilliant Green.

In another aspect of this embodiment, a pre-enrichment media comprises4.7 g/L Bile Salts, 4.3 g/L Meat Extract, 8.6 g/L Caseine Peptone, 2.6g/L NaCl, 38.7 g/L, CaCO₃, 30.5 g/L, Na₂S₂O₃, 4 g/L Iodine, 4 g/LPotassium Iodide, 0.008 g/L Novobiocin, and 0.008 g/L Brilliant Green.

In aspects of this embodiment, a pre-enrichment media comprises 7 g/L to11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5g/L Meat Extract, 3 g/L to 5 g/L of a first iodine compound, 3 g/L to 5g/L of a second iodine compound, 0.002 g/L to 0.006 g/L of ananti-microbial compound, 0.002 g/L to 0.006 g/L of a triarylmethane dye.In other aspects of this embodiment, a pre-enrichment media comprises 7g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/Lto 5 g/L Meat Extract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L ofPotassium Iodide, 0.002 g/L to 0.006 g/L of an aminocoumarin antibiotic,0.002 g/L to 0.006 g/L of a Malachite green dye. In yet other aspects ofthis embodiment, a pre-enrichment media comprises 7 g/L to 11 g/L of aCaseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5 g/L MeatExtract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide,0.002 g/L to 0.006 g/L of Novobiocin, 0.002 g/L to 0.006 g/L ofBrilliant Green.

In another aspect of this embodiment, a pre-enrichment media comprises4.7 g/L Bile Salts, 4.3 g/L Meat Extract, 8.6 g/L Caseine Peptone, 2.6g/L NaCl, 38.7 g/L, CaCO₃, 30.5 g/L, Na₂S₂O₃, 4 g/L Iodine, 4 g/LPotassium Iodide, 0.008 g/L Novobiocin, and 0.008 g/L Brilliant Green.In another aspect of this embodiment, a pre-enrichment media comprises4.7 g/L Bile Salts, 4.3 g/L Meat Extract, 8.6 g/L Caseine Peptone, 2.6g/L NaCl, 38.7 g/L, CaCO₃, 30.5 g/L, Na₂S₂O₃, 4 g/L Iodine, 4 g/LPotassium Iodide, 0.004 g/L Novobiocin, and 0.004 g/L Brilliant Green.In another aspect of this embodiment, a pre-enrichment media comprises2.4 g/L Bile Salts, 2.1 g/L Meat Extract, 4.3 g/L Caseine Peptone, 1.3g/L NaCl, 19.3 g/L, CaCO₃, 15.2 g/L, Na₂S₂O₃, 2 g/L Iodine, 2 g/LPotassium Iodide, 0.004 g/L Novobiocin, and 0.004 g/L Brilliant Green.In another aspect of this embodiment, a pre-enrichment media comprises2.4 g/L Bile Salts, 4.3 g/L Soy Peptone, 1.3 g/L NaCl, 19.3 g/L, CaCO₃,15.2 g/L, Na₂S₂O₃, 2 g/L Iodine, 2 g/L Potassium Iodide, 0.004 g/LNovobiocin, and 0.004 g/L Brilliant Green.

In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of apeptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of a first growthinhibiting agent, 2 g/L to 6 g/L of a second growth inhibiting agent,and 0.001 g/L to 0.0012 g/L of a third growth inhibiting agent. Inaspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15g/L of a Caseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/Lof a first iodine compound, 2 g/L to 6 g/L of a second iodine compound,and 0.001 g/L to 0.0012 g/L of an anti-microbial compound. In otheraspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15g/L of a Caseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/Lof a first iodine compound, 2 g/L to 6 g/L of a second iodine compound,and 0.001 g/L to 0.0012 g/L of a triarylmethane dye.

In aspects of this embodiment, a pre-enrichment media comprises 8 g/L to12 g/L of a Caseine peptone, 3 g/L to 5 g/L Bile Salts, 3 g/L to 5 g/Lof a first iodine compound, 3 g/L to 5 g/L of a second iodine compound,and 0.002 g/L to 0.006 g/L of an anti-microbial compound. In otheraspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12of a Caseine peptone, 3 g/L to 5 g/L Bile Salts, 3 g/L to 5 g/L ofIodine, 3 g/L to 5 g/L of Potassium Iodide, and 0.002 g/L to 0.006 g/Lof an aminocoumarin antibiotic. In yet other aspects of this embodiment,a pre-enrichment media comprises 8 g/L to 12 of a Caseine peptone, 3 g/Lto 5 g/L Bile Salts, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L ofPotassium Iodide, and 0.002 g/L to 0.006 g/L of Novobiocin.

In another aspect of this embodiment, a pre-enrichment media comprises10 g/L Caseine Peptone, 4 g/L Bile Salts, 3.5 g/L Disodium Phosphate,1.5 g/L Potassium Phosphate, 7.6 g/L NaCl, 30.5 g/L, Na₂S₂O₃, 4 g/LIodide, 4 g/L Potassium Iodide, and 0.004 g/L Novobiocin. In anotheraspect of this embodiment, a pre-enrichment media comprises 10 g/LCaseine Peptone, 2.4 g/L Bile Salts, 3.5 g/L Disodium Phosphate, 1.5 g/LPotassium Phosphate, 6.3 g/L NaCl, 15.2 g/L, Na₂S₂O₃, 4 g/L Iodide, 4g/L Potassium Iodide, 0.004 g/L Novobiocin, In another aspect of thisembodiment, a pre-enrichment media comprises 10 g/L Caseine Peptone, 2.4g/L Bile Salts, 3.5 g/L Disodium Phosphate, 1.5 g/L Potassium Phosphate,6.3 g/L NaCl, 19.3 g/L, CaCO₃, 15.2 g/L, Na₂S₂O₃, 4 g/L Iodide, 4 g/LPotassium Iodide, and 0.004 g/L Novobiocin.

In an embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of aTryptone and 0.5 g/L to 3 g/L of a growth inhibiting agent. In aspectsof this embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of aTryptone and 0.5 g/L to 3 g/L of an anti-microbial compound. In otheraspects of this embodiment, a pre-enrichment media comprises 3 g/L to 7g/L of a Tryptone and 0.5 g/L to 3 g/L of a selenium compound. In yetother aspects of this embodiment, a pre-enrichment media comprises 3 g/Lto 7 g/L of a Tryptone and 0.5 g/L to 3 g/L of Sodium Hydrogen Selenite.In still other aspects of this embodiment, a pre-enrichment mediacomprises 4 g/L to 6 g/L of a Tryptone and 1 g/L to 2 g/L of SodiumHydrogen Selenite. In another aspect of this embodiment, apre-enrichment media comprises 5 g/L Tryptone, 4 g/L Lactose, 10 g/LDisodium Phosphate, 0.01 g/L L-Cystine, and 1.51 g/L Sodium HydrogenSelenite.

In an embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of aTryptone, 1 g/L to 3 g/L of Bile Salts, 2 g/L to 6 g/L of a first growthinhibiting agent, 2 g/L to 6 g/L of a second growth inhibiting agent,and 0.001 g/L to 0.0012 g/L of a third growth inhibiting agent. Inaspects of this embodiment, a pre-enrichment media comprises 3 g/L to 7g/L of a Tryptone, 1 g/L to 3 g/L of Bile Salts, 2 g/L to 6 g/L of afirst iodine compound, 2 g/L to 6 g/L of a second iodine compound, and0.001 g/L to 0.0012 g/L of an anti-microbial compound. In other aspectsof this embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of aTryptone, 1 g/L to 3 g/L of Bile Salts, 2 g/L to 6 g/L of a first iodinecompound, 2 g/L to 6 g/L of a second iodine compound, and 0.001 g/L to0.0012 g/L of a triarylmethane dye.

In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to6 g/L of a Tryptone, 1.5 g/L to 3.5 g/L Bile Salts, 3 g/L to 5 g/L of afirst iodine compound, 3 g/L to 5 g/L of a second iodine compound, and0.002 g/L to 0.006 g/L of an anti-microbial compound. In other aspectsof this embodiment, a pre-enrichment media comprises 4 g/L to 6 g/L of aTryptone, 1.5 g/L to 3.5 g/L Bile Salts, 3 g/L to 5 g/L of Iodine, 3 g/Lto 5 g/L of Potassium Iodide, and 0.002 g/L to 0.006 g/L of anaminocoumarin antibiotic. In yet other aspects of this embodiment, apre-enrichment media comprises 4 g/L to 6 g/L of a Tryptone, 1.5 g/L to3.5 g/L Bile Salts, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L ofPotassium Iodide, and 0.002 g/L to 0.006 g/L of Novobiocin.

In another aspect of this embodiment, a pre-enrichment media comprises 5g/L Tryptone, 4 g/L Lactose, 2.4 g/L Bile Salts, 10 g/L DisodiumPhosphate, 0.01 g/L L-Cystine, 19.3 g/L, CaCO₃, 30.5 g/L, Na₂S₂O₃, 4 g/LIodide, 4 g/L Potassium Iodide, and 0.004 g/L Novobiocin. In anotheraspect of this embodiment, a pre-enrichment media comprises 5 g/LTryptone, 4 g/L Lactose, 2.4 g/L Bile Salts, 10 g/L Disodium Phosphate,0.01 g/L L-Cystine, 30.5 g/L, Na₂S₂O₃, 4 g/L Iodide, 4 g/L PotassiumIodide, and 0.004 g/L Novobiocin.

Aspect of the present specification disclose, in part, incubation of asample in a pre-enrichment media. Incubation of a sample is performedunder temperature and time parameters that facilitate the growth of thepathogen in the sample, retard the growth of an unwanted organism in thesample, and/or establish conditions that otherwise increase thepopulation of the pathogen in the sample and/or retard the growth of anunwanted organism in the sample. Incubation of pre-enrichment media maybe performed under constant rotation, reversal or agitation.

Any temperature may be used during incubation of a sample in apre-enrichment media, with the proviso that the temperature is useful topractice the methods disclosed herein. In aspects of this embodiment, atemperature used to incubate of a sample in a pre-enrichment media maybe, e.g., about 25° C., about 26° C., about 27° C., about 28° C., about29° C., about 30° C., about 31° C., about 32° C., about 33° C., about34° C., about 35° C., about 36° C., about 37° C., about 38° C., about39° C., about 40° C., about 41° C., or about 42° C. In other aspects ofthis embodiment, a temperature used to incubate of a sample in apre-enrichment media may be, e.g., at least 25° C., at least 26° C., atleast 27° C., at least 28° C., at least 29° C., at least 30° C., atleast 31° C., at least 32° C., at least 33° C., at least 34° C., atleast 35° C., at least 36° C., at least 37° C., at least 38° C., atleast 39° C., at least 40° C., at least 41° C., or at least 42° C. Inyet other aspects of this embodiment, a temperature used to incubate ofa sample in a pre-enrichment media may be, e.g., at most 25° C., at most26° C., at most 27° C., at most 28° C., at most 29° C., at most 30° C.,at most 31° C., at most 32° C., at most 33° C., at most 34° C., at most35° C., at most 36° C., at most 37° C., at most 38° C., at most 39° C.,at most 40° C., at most 41° C., or at most 42° C. In still other aspectsof this embodiment, a temperature used to incubate of a sample in apre-enrichment media may be, e.g., about 25° C. to about 29° C., about26° C. to about 30° C., about 27° C. to about 31° C., about 28° C. toabout 32° C., about 29° C. to about 33° C., about 30° C. to about 34°C., about 31° C. to about 35° C., about 32° C. to about 36° C., about33° C. to about 37° C., about 34° C. to about 38° C., about 35° C. toabout 39° C., about 36° C. to about 40° C., about 37° C. to about 41°C., about 38° C. to about 42° C., about 39° C. to about 43° C., or about40° C. to about 44° C.

Any time may be used during incubation of a sample in a pre-enrichmentmedia, with the proviso that the time is useful to practice the methodsdisclosed herein. In aspects of this embodiment, a time used to incubateof a sample in a pre-enrichment media may be, e.g., about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, or about 24 hours. In other aspects of this embodiment, a timeused to incubate of a sample in a pre-enrichment media may be, e.g., atleast 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, atleast 8 hours, at least 9 hours, at least 10 hours, at least 11 hours,at least 12 hours, at least 13 hours, at least 14 hours, at least 15hours, at least 16 hours, at least 17 hours, at least 18 hours, at least19 hours, at least 20 hours, at least 21 hours, at least 22 hours, atleast 23 hours, or at least 24 hours. In yet other aspects of thisembodiment, a time used to incubate of a sample in a pre-enrichmentmedia may be, e.g., at most 4 hours, at most 5 hours, at most 6 hours,at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, atmost 11 hours, at most 12 hours, at most 13 hours, at most 14 hours, atmost 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, atmost 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, atmost 23 hours, or at most 24 hours. In yet other aspects of thisembodiment, a time used to incubate of a sample in a pre-enrichmentmedia may be, e.g., about 4 hours to about 6 hours, about 5 hours toabout 7 hours, about 6 hours to about 8 hours, about 7 hours to about 9hours, about 8 hours to about 10 hours, about 9 hours to about 11 hours,about 10 hours to about 12 hours, about 11 hours to about 13 hours,about 12 hours to about 14 hours, about 13 hours to about 15 hours,about 14 hours to about 16 hours, about 15 hours to about 17 hours,about 16 hours to about 18 hours, about 17 hours to about 19 hours,about 18 hours to about 20 hours, about 19 hours to about 21 hours,about 20 hours to about 22 hours, about 21 hours to about 23 hours,about 22 hours to about 24 hours, about 23 hours to about 25 hours, orabout 24 hours to about 26 hours.

In aspects of this embodiment, a sample in a pre-enrichment media may beincubated at a temperature of, e.g., about 25° C., about 26° C., about27° C., about 28° C., about 29° C., about 30° C., about 31° C., about32° C., about 33° C., about 34° C., about 35° C., about 36° C., about37° C., about 38° C., about 39° C., about 40° C., about 41° C., or about42° C. for a time of, e.g., about 4 hours, about 5 hours, about 6 hours,about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours,about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20hours, about 21 hours, about 22 hours, about 23 hours, or about 24hours. Incubation of pre-enrichment media may be performed underconstant rotation, reversal or agitation.

In other aspects of this embodiment, a sample in a pre-enrichment mediamay be incubated at a temperature of, e.g., at least 25° C., at least26° C., at least 27° C., at least 28° C., at least 29° C., at least 30°C., at least 31° C., at least 32° C., at least 33° C., at least 34° C.,at least 35° C., at least 36° C., at least 37° C., at least 38° C., atleast 39° C., at least 40° C., at least 41° C., or at least 42° C. for atime of, e.g., at least 4 hours, at least 5 hours, at least 6 hours, atleast 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, atleast 11 hours, at least 12 hours, at least 13 hours, at least 14 hours,at least 15 hours, at least 16 hours, at least 17 hours, at least 18hours, at least 19 hours, at least 20 hours, at least 21 hours, at least22 hours, at least 23 hours, or at least 24 hours. Incubation ofpre-enrichment media may be performed under constant rotation, reversalor agitation.

In yet other aspects of this embodiment, a sample in a pre-enrichmentmedia may be incubated at a temperature of, e.g., at most 25° C., atmost 26° C., at most 27° C., at most 28° C., at most 29° C., at most 30°C., at most 31° C., at most 32° C., at most 33° C., at most 34° C., atmost 35° C., at most 36° C., at most 37° C., at most 38° C., at most 39°C., at most 40° C., at most 41° C., or at most 42° C. for a time of,e.g., at most 4 hours, at most 5 hours, at most 6 hours, at most 7hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23hours, or at most 24 hours. Incubation of pre-enrichment media may beperformed under constant rotation, reversal or agitation.

In still other aspects of this embodiment, a sample in a pre-enrichmentmedia may be incubated at a temperature of, e.g., about 25° C. to about29° C., about 26° C. to about 30° C., about 27° C. to about 31° C.,about 28° C. to about 32° C., about 29° C. to about 33° C., about 30° C.to about 34° C., about 31° C. to about 35° C., about 32° C. to about 36°C., about 33° C. to about 37° C., about 34° C. to about 38° C., about35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. toabout 41° C., about 38° C. to about 42° C., about 39° C. to about 43°C., or about 40° C. to about 44° C. for a time of, e.g., about 4 hoursto about 6 hours, about 5 hours to about 7 hours, about 6 hours to about8 hours, about 7 hours to about 9 hours, about 8 hours to about 10hours, about 9 hours to about 11 hours, about 10 hours to about 12hours, about 11 hours to about 13 hours, about 12 hours to about 14hours, about 13 hours to about 15 hours, about 14 hours to about 16hours, about 15 hours to about 17 hours, about 16 hours to about 18hours, about 17 hours to about 19 hours, about 18 hours to about 20hours, about 19 hours to about 21 hours, about 20 hours to about 22hours, about 21 hours to about 23 hours, about 22 hours to about 24hours, about 23 hours to about 25 hours, or about 24 hours to about 26hours. Incubation of pre-enrichment media may be performed underconstant rotation, reversal or agitation.

A method for the detection of a pathogen in a sample comprises the stepof incubation of the sample in an enrichment media. After apre-enrichment media step disclosed herein is completed, an aliquot ofthe pre-enrichment media is transferred to an enrichment media forsubsequent growth of the pathogen. An enrichment step comprisesincubating an aliquot of the pre-enrichment media in an enrichment mediafor a defined time and at a defined temperature.

Any volume of an aliquoit of pre-enrichment media may be used duringincubation of a pre-enrichment media in a enrichment media, with theproviso that the volume is useful to practice the methods disclosedherein. In aspects of this embodiment, an aliquot volume ofpre-enrichment media transferred to an enrichment media may be, e.g.,about 1/50, about 1/75, about 1/100, about 1/125, about 1/150, about1/175, about 1/200, about 1/225, about 1/250, about 1/275, about 1/300,about 1/325, about 1/350, about 1/375, about 1/400, about 1/425, about1/450, about 1/475, about 1/500, about 1/525, about 1/550, about 1/575,about 1/600, about 1/625, about 1/650, about 1/675, about 1/700, about1/725, about 1/750, about 1/775, about 1/800, about 1/825, about 1/850,about 1/875, about 1/900, about 1/925, about 1/950, about 1/975, orabout 1/1,000 the volume of an enrichment media used in an enrichmentstep. In other aspects of this embodiment, an aliquot volume ofpre-enrichment media transferred to an enrichment media may be, e.g., atleast 1/50, at least 1/75, at least 1/100, at least 1/125, at least1/150, at least 1/175, at least 1/200, at least 1/225, at least 1/250,at least 1/275, at least 1/300, at least 1/325, at least 1/350, at least1/375, at least 1/400, at least 1/425, at least 1/450, at least 1/475,at least 1/500, at least 1/525, at least 1/550, at least 1/575, at least1/600, at least 1/625, at least 1/650, at least 1/675, at least 1/700,at least 1/725, at least 1/750, at least 1/775, at least 1/800, at least1/825, at least 1/850, at least 1/875, at least 1/900, at least 1/925,at least 1/950, at least 1/975, or at least 1/1,000 the volume of anenrichment media used in an enrichment step. In yet other aspects ofthis embodiment, an aliquot volume of pre-enrichment media transferredto an enrichment media may be, e.g., at most 1/50, at most 1/75, at most1/100, at most 1/125, at most 1/150, at most 1/175, at most 1/200, atmost 1/225, at most 1/250, at most 1/275, at most 1/300, at most 1/325,at most 1/350, at most 1/375, at most 1/400, at most 1/425, at most1/450, at most 1/475, at most 1/500, at most 1/525, at most 1/550, atmost 1/575, at most 1/600, at most 1/625, at most 1/650, at most 1/675,at most 1/700, at most 1/725, at most 1/750, at most 1/775, at most1/800, at most 1/825, at most 1/850, at most 1/875, at most 1/900, atmost 1/925, at most 1/950, at most 1/975, or at most 1/1,000 the volumeof an enrichment media used in an enrichment step.

In yet other aspects of this embodiment, an aliquot volume ofpre-enrichment media transferred to an enrichment media may be, e.g.,about 1/50 to about 1/100, about 1/50 to about 1/150, about 1/50 toabout 1/200, about 1/50 to about 1/250, about 1/50 to about 1/300, about1/50 to about 1/350, about 1/50 to about 1/400, about 1/50 to about1/450, about 1/50 to about 1/500, about 1/550 to about 1/600, about 1/50to about 1/650, about 1/50 to about 1/700, about 1/50 to about 1/750,about 1/50 to about 1/800, about 1/50 to about 1/850, about 1/50 toabout 1/900, about 1/50 to about 1/950, about 1/50 to about 1/1,000,about 1/100 to about 1/150, about 1/100 to about 1/200, about 1/100 toabout 1/250, about 1/100 to about 1/300, about 1/100 to about 1/350,about 1/100 to about 1/400, about 1/100 to about 1/450, about 1/100 toabout 1/500, about 1/100 to about 1/550, about 1/100 to about 1/600,about 1/100 to about 1/650, about 1/100 to about 1/700, about 1/100 toabout 1/750, about 1/100 to about 1/800, about 1/100 to about 1/850,about 1/100 to about 1/900, about 1/100 to about 1/950, about 1/100 toabout 1/1,000, about 1/200 to about 1/250, about 1/200 to about 1/300,about 1/200 to about 1/350, about 1/200 to about 1/400, about 1/200 toabout 1/450, about 1/200 to about 1/500, about 1/200 to about 1/550,about 1/200 to about 1/600, about 1/200 to about 1/650, about 1/200 toabout 1/700, about 1/200 to about 1/750, about 1/200 to about 1/800,about 1/200 to about 1/850, about 1/200 to about 1/900, about 1/200 toabout 1/950, about 1/200 to about 1/1,000, about 1/300 to about 1/350,about 1/300 to about 1/400, about 1/300 to about 1/450, about 1/300 toabout 1/500, about 1/300 to about 1/550, about 1/300 to about 1/600,about 1/300 to about 1/650, about 1/300 to about 1/700, about 1/300 toabout 1/750, about 1/300 to about 1/800, about 1/300 to about 1/850,about 1/300 to about 1/900, about 1/300 to about 1/950, about 1/300 toabout 1/1,000, about 1/400 to about 1/450, about 1/400 to about 1/500,about 1/400 to about 1/550, about 1/400 to about 1/600, about 1/400 toabout 1/650, about 1/400 to about 1/700, about 1/400 to about 1/750,about 1/400 to about 1/800, about 1/400 to about 1/850, about 1/400 toabout 1/900, about 1/400 to about 1/950, about 1/400 to about 1/1,000,about 1/500 to about 1/550, about 1/500 to about 1/600, about 1/500 toabout 1/650, about 1/500 to about 1/700, about 1/500 to about 1/750,about 1/500 to about 1/800, about 1/500 to about 1/850, about 1/500 toabout 1/900, about 1/500 to about 1/950, or about 1/500 to about1/1,000.

In aspects of this embodiment, a ratio of pre-enrichment media toenrichment media may used in an enrichment step may be, e.g., about1:50, about 1:75, about 1:100, about 1:125, about 1:150, about 1:175,about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about1:325, about 1:350, about 1:375, about 1:400, about 1:425, about 1:450,about 1:475, about 1:500, about 1:525, about 1:550, about 1:575, about1:600, about 1:625, about 1:650, about 1:675, about 1:700, about 1:725,about 1:750, about 1:775, about 1:800, about 1:825, about 1:850, about1:875, about 1:900, about 1:925, about 1:950, about 1:975, or about1:1,000. In other aspects of this embodiment, a ratio of pre-enrichmentmedia to enrichment media may used in an enrichment step may be, e.g.,at least 1:50, at least 1:75, at least 1:100, at least 1:125, at least1:150, at least 1:175, at least 1:200, at least 1:225, at least 1:250,at least 1:275, at least 1:300, at least 1:325, at least 1:350, at least1:375, at least 1:400, at least 1:425, at least 1:450, at least 1:475,at least 1:500, at least 1:525, at least 1:550, at least 1:575, at least1:600, at least 1:625, at least 1:650, at least 1:675, at least 1:700,at least 1:725, at least 1:750, at least 1:775, at least 1:800, at least1:825, at least 1:850, at least 1:875, at least 1:900, at least 1:925,at least 1:950, at least 1:975, or at least 1:1,000. In yet otheraspects of this embodiment, a ratio of pre-enrichment media toenrichment media may used in an enrichment step may be, e.g., at most1:50, at most 1:75, at most 1:100, at most 1:125, at most 1:150, at most1:175, at most 1:200, at most 1:225, at most 1:250, at most 1:275, atmost 1:300, at most 1:325, at most 1:350, at most 1:375, at most 1:400,at most 1:425, at most 1:450, at most 1:475, at most 1:500, at most1:525, at most 1:550, at most 1:575, at most 1:600, at most 1:625, atmost 1:650, at most 1:675, at most 1:700, at most 1:725, at most 1:750,at most 1:775, at most 1:800, at most 1:825, at most 1:850, at most1:875, at most 1:900, at most 1:925, at most 1:950, at most 1:975, or atmost 1:1,000.

In still other aspects of this embodiment, a ratio of pre-enrichmentmedia to enrichment media may used in an enrichment step may be, e.g.,about 1:50 to about 1:100, about 1:50 to about 1:150, about 1:50 toabout 1:200, about 1:50 to about 1:250, about 1:50 to about 1:300, about1:50 to about 1:350, about 1:50 to about 1:400, about 1:50 to about1:450, about 1:50 to about 1:500, about 1:50 to about 1:550, about 1:50to about 1:600, about 1:50 to about 1:650, about 1:50 to about 1:700,about 1:50 to about 1:750, about 1:50 to about 1:800, about 1:50 toabout 1:850, about 1:50 to about 1:900, about 1:50 to about 1:950, about1:50 to about 1:1,000, about 1:100 to about 1:200, about 1:100 to about1:250, about 1:100 to about 1:300, about 1:100 to about 1:350, about1:100 to about 1:400, about 1:100 to about 1:450, about 1:100 to about1:500, about 1:100 to about 1:550, about 1:100 to about 1:600, about1:100 to about 1:650, about 1:100 to about 1:700, about 1:100 to about1:750, about 1:100 to about 1:800, about 1:100 to about 1:850, about1:100 to about 1:900, about 1:100 to about 1:950, about 1:100 to about1:1,000, about 1:200 to about 1:250, about 1:200 to about 1:300, about1:200 to about 1:350, about 1:200 to about 1:400, about 1:200 to about1:450, about 1:200 to about 1:500, about 1:200 to about 1:550, about1:200 to about 1:600, about 1:200 to about 1:650, about 1:200 to about1:700, about 1:200 to about 1:750, about 1:200 to about 1:800, about1:200 to about 1:850, about 1:200 to about 1:900, about 1:200 to about1:950, about 1:200 to about 1:1,000, about 1:300 to about 1:350, about1:300 to about 1:400, about 1:300 to about 1:450, about 1:300 to about1:500, about 1:300 to about 1:550, about 1:300 to about 1:600, about1:300 to about 1:650, about 1:300 to about 1:700, about 1:300 to about1:750, about 1:300 to about 1:800, about 1:300 to about 1:850, about1:300 to about 1:900, about 1:300 to about 1:950, about 1:300 to about1:1,000, about 1:400 to about 1:450, about 1:400 to about 1:500, about1:400 to about 1:550, about 1:400 to about 1:600, about 1:400 to about1:650, about 1:400 to about 1:700, about 1:400 to about 1:750, about1:400 to about 1:800, about 1:400 to about 1:850, about 1:400 to about1:900, about 1:400 to about 1:950, about 1:400 to about 1:1,000, about1:500 to about 1:550, about 1:500 to about 1:600, about 1:500 to about1:650, about 1:500 to about 1:700, about 1:500 to about 1:750, about1:500 to about 1:800, about 1:500 to about 1:850, about 1:500 to about1:900, about 1:500 to about 1:950, about 1:500 to about 1:1,000, about1:600 to about 1:650, about 1:600 to about 1:700, about 1:600 to about1:750, about 1:600 to about 1:800, about 1:600 to about 1:850, about1:600 to about 1:900, about 1:600 to about 1:950, about 1:600 to about1:1,000, about 1:700 to about 1:750, about 1:700 to about 1:800, about1:700 to about 1:850, about 1:700 to about 1:900, about 1:700 to about1:950, about 1:700 to about 1:1,000, about 1:800 to about 1:850, about1:800 to about 1:900, about 1:800 to about 1:950, about 1:800 to about1:1,000, about 1:900 to about 1:950, about 1:900 to about 1:1,000, orabout 1:950 to about 1:1,000.

Aspect of the present specification disclose, in part, an enrichmentmedia. An enrichment media, also referred to as an enrichment culturemedia is a buffered culture media that provides the nutrients necessaryto sustain high-growth of the pathogen. This is typically done bytailoring the media particularly conducive to the growth of the pathogensuch as, e.g., considering survivable osmotic pressure ranges,survivable pH ranges, resistance to selective compounds, minimalnutritional requirements. Non-limiting examples of an enrichment mediainclude a Rappaport Vassiliadis Soya Medium (RVS), a McConkey Broth, aFraser Broth, a Soy-Triptone Broth (TSB), a Reinforce Clostridium Broth,a Campylobacter Thioglycolate Medium, a Nitrate Broth, a Triple SugarIron Broth (TSI), a Sodium Hippurate Broth, a Selenite Cystine Broth, aGN Broth, a Todd Hewitt Broth, a Malt Extract Broth, an Azide DextroseBroth, and a Hektoen Broth.

In an aspect of this embodiment, when the pathogen is a Salmonella sp.,the enrichment media may be a Rappaport Vassiliadis Soya Medium, aSelenite Cystine Broth, or a GN Broth. In another aspect of thisembodiment, when the pathogen is an Escherichia coli, the enrichmentmedia may be a McConkey Broth. In yet another aspect of this embodiment,when the pathogen is a Listeria monocytogenes, the enrichment media maybe a Fraser Broth. In still another aspect of this embodiment, when thepathogen is a Staphylococcus aureus, the enrichment media may be aSoy-Triptone Broth (TSB). In another aspect of this embodiment, when thepathogen is a Clostridium sp., the enrichment media may be a ReinforceClostridium Broth. In yet another aspect of this embodiment, when thepathogen is a Campylobacter sp., the enrichment media may be aCampylobacter Thioglycolate Medium.

In another aspect of this embodiment, when the pathogen is a Pseudomonassp., the enrichment media may be a Nitrate Broth or a Triple Sugar IronBroth (TSI). In yet another aspect of this embodiment, when the pathogenis an Acinetobacter sp., the enrichment media may be a Nitrate Broth ora Triple Sugar Iron Broth (TSI). In still another aspect of thisembodiment, when the pathogen is a Legionella sp., the enrichment mediamay be a Sodium Hippurate Broth. In another aspect of this embodiment,when the pathogen is a Shigella sp., the enrichment media may be aSelenite Cystine Broth or a GN Broth. In yet another aspect of thisembodiment, when the pathogen is a Streptococcus sp., the enrichmentmedia may be a Todd Hewitt Broth. In another aspect of this embodiment,when the pathogen is an Enterococcus sp., the enrichment media may be anAzide Dextrose Broth.

In another aspect of this embodiment, when the pathogen is a fungus, theenrichment media may be a Malt Extract Broth. In yet another aspect ofthis embodiment, when the pathogen is a yeast, the enrichment media maybe a Malt Extract Broth. In still another aspect of this embodiment,when the pathogen is a mold, the enrichment media may be a Malt ExtractBroth. In another aspect of this embodiment, when the pathogen is aProteus, the enrichment media may be a Hektoen Broth.

An enrichment media typically comprises a high growth nutrient componentused as a source of proteins, amino acids and nitrogen. Either a singlehigh growth nutrient component may comprise an enrichment mediadisclosed herein, or a plurality of high growth nutrient components maycomprise an enrichment media disclosed herein. A non-limiting example ofa high growth nutrient component is a peptone as disclosed herein.

Any concentration of high growth nutrient component may be used, withthe proviso that the concentration is useful to practice the methodsdisclosed herein. In aspects of this embodiment, a high growth nutrientcomponent may be used at a concentration of, e.g., about 1 g/L, about 2g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L,about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L,about 13 g/L, about 14 g/L, or about 15 g/L. In other aspects of thisembodiment, a high growth nutrient component may be used at aconcentration of, e.g., at least 1 g/L, at least 2 g/L, at least 3 g/L,at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12g/L, at least 13 g/L, at least 14 g/L, or at least 15 g/L. In yet otheraspects of this embodiment, a high growth nutrient component may be usedat a concentration of, e.g., at most 1 g/L, at most 2 g/L, at most 3g/L, at most 4 g/L, at most 5 g/L, at most 6 g/L, at most 7 g/L, at most8 g/L, at most 9 g/L, at most 10 g/L, at most 11 g/L, at most 12 g/L, atmost 13 g/L, at most 14 g/L, or at most 15 g/L.

In yet other aspects of this embodiment, a high growth nutrientcomponent may be used at a concentration of between, e.g., about 1 g/Lto 2 g/L, about 1 g/L to 3 g/L, about 1 g/L to 4 g/L, about 1 g/L to 5g/L, about 1 g/L to 6 g/L, about 1 g/L to 7 g/L, about 1 g/L to 8 g/L,about 1 g/L to 9 g/L, about 1 g/L to 10 g/L, about 1 g/L to 11 g/L,about 1 g/L to 12 g/L, about 1 g/L to 13 g/L, about 1 g/L to 14 g/L,about 1 g/L to 15 g/L, about 2 g/L to 3 g/L, about 2 g/L to 4 g/L, about2 g/L to 5 g/L, about 2 g/L to 6 g/L, about 2 g/L to 7 g/L, about 2 g/Lto 8 g/L, about 2 g/L to 9 g/L, about 2 g/L to 10 g/L, about 2 g/L to 11g/L, about 2 g/L to 12 g/L, about 2 g/L to 13 g/L, about 2 g/L to 14g/L, about 2 g/L to 15 g/L, about 3 g/L to 4 g/L, about 3 g/L to 5 g/L,about 3 g/L to 6 g/L, about 3 g/L to 7 g/L, about 3 g/L to 8 g/L, about3 g/L to 9 g/L, about 3 g/L to 10 g/L, about 3 g/L to 11 g/L, about 3g/L to 12 g/L, about 3 g/L to 13 g/L, about 3 g/L to 14 g/L, about 3 g/Lto 15 g/L, about 4 g/L to 5 g/L, about 4 g/L to 6 g/L, about 4 g/L to 7g/L, about 4 g/L to 8 g/L, about 4 g/L to 9 g/L, about 4 g/L to 10 g/L,about 4 g/L to 11 g/L, about 4 g/L to 12 g/L, about 4 g/L to 13 g/L,about 4 g/L to 14 g/L, about 4 g/L to 15 g/L, about 5 g/L to 6 g/L,about 5 g/L to 7 g/L, about 5 g/L to 8 g/L, about 5 g/L to 9 g/L, about5 g/L to 10 g/L, about 5 g/L to 11 g/L, about 5 g/L to 12 g/L, about 5g/L to 13 g/L, about 5 g/L to 14 g/L, about 5 g/L to 15 g/L, about 6 g/Lto 7 g/L, about 6 g/L to 8 g/L, about 6 g/L to 9 g/L, about 6 g/L to 10g/L, about 6 g/L to 11 g/L, about 6 g/L to 12 g/L, about 6 g/L to 13g/L, about 6 g/L to 14 g/L, about 6 g/L to 15 g/L, about 7 g/L to 8 g/L,about 7 g/L to 9 g/L, about 7 g/L to 10 g/L, about 7 g/L to 11 g/L,about 7 g/L to 12 g/L, about 7 g/L to 13 g/L, about 7 g/L to 14 g/L,about 7 g/L to 15 g/L, about 8 g/L to 9 g/L, about 8 g/L to 10 g/L,about 8 g/L to 11 g/L, about 8 g/L to 12 g/L, about 8 g/L to 13 g/L,about 8 g/L to 14 g/L, about 8 g/L to 15 g/L, about 9 g/L to 10 g/L,about 9 g/L to 11 g/L, about 9 g/L to 12 g/L, about 9 g/L to 13 g/L,about 9 g/L to 14 g/L, about 9 g/L to 15 g/L, about 10 g/L to 11 g/L,about 10 g/L to 12 g/L, about 10 g/L to 13 g/L, about 10 g/L to 14 g/L,about 10 g/L to 15 g/L, about 11 g/L to 12 g/L, about 11 g/L to 13 g/L,about 11 g/L to 14 g/L, about 11 g/L to 15 g/L, about 12 g/L to 13 g/L,about 12 g/L to 14 g/L, about 12 g/L to 15 g/L, about 13 g/L to 14 g/L,about 13 g/L to 15 g/L, or about 14 g/L to 15 g/L.

An enrichment media typically comprises a growth promoting agent.Non-limiting examples of a growth promoting agent is an iron containingcompound that can be used by a pathogen of interest as an iron source.In an aspect of this embodiment, a growth promoting agent is ammoniumferric citrate.

Any concentration of ammonium ferric citrate may be used, with theproviso that the concentration is useful to practice the methodsdisclosed herein. In aspects of this embodiment, ammonium ferric citratemay be used at a concentration of, e.g., about 0.01 mg/mL, about 0.02mg/mL, about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9mg/mL, about 1.0 mg/mL, about 2.0 mg/mL, about 3.0 mg/mL, about 4.0mg/mL, about 5.0 mg/mL, about 6.0 mg/mL, about 7.0 mg/mL, about 8.0mg/mL, about 9.0 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL,about 13 mg/mL, about 14 mg/mL, or about 15 mg/mL. In other aspects ofthis embodiment, ammonium ferric citrate may be used at a concentrationof, e.g., at least 0.01 mg/mL, at least 0.02 mg/mL, at least 0.03 mg/mL,at least 0.04 mg/mL, at least 0.05 mg/mL, at least 0.06 mg/mL, at least0.07 mg/mL, at least 0.08 mg/mL, at least 0.09 mg/mL, at least 0.1mg/mL, at least 0.2 mg/mL, at least 0.3 mg/mL, at least 0.4 mg/mL, atleast 0.5 mg/mL, at least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8mg/mL, at least 0.9 mg/mL, at least 1.0 mg/mL, at least 2.0 mg/mL, atleast 3.0 mg/mL, at least 4.0 mg/mL, at least 5.0 mg/mL, at least 6.0mg/mL, at least 7.0 mg/mL, at least 8.0 mg/mL, at least 9.0 mg/mL, atleast 10 mg/mL, at least 11 mg/mL, at least 12 mg/mL, at least 13 mg/mL,at least 14 mg/mL, or at least 15 mg/mL. In yet other aspects of thisembodiment, ammonium ferric citrate may be used at a concentration of,e.g., at most 0.01 mg/mL, at most 0.02 mg/mL, at most 0.03 mg/mL, atmost 0.04 mg/mL, at most 0.05 mg/mL, at most 0.06 mg/mL, at most 0.07mg/mL, at most 0.08 mg/mL, at most 0.09 mg/mL, at most 0.1 mg/mL, atmost 0.2 mg/mL, at most 0.3 mg/mL, at most 0.4 mg/mL, at most 0.5 mg/mL,at most 0.6 mg/mL, at most 0.7 mg/mL, at most 0.8 mg/mL, at most 0.9mg/mL, at most 1.0 mg/mL, at most 2.0 mg/mL, at most 3.0 mg/mL, at most4.0 mg/mL, at most 5.0 mg/mL, at most 6.0 mg/mL, at most 7.0 mg/mL, atmost 8.0 mg/mL, at most 9.0 mg/mL, at most 10 mg/mL, at most 11 mg/mL,at most 12 mg/mL, at most 13 mg/mL, at most 14 mg/mL, or at most 15mg/mL.

In still other aspects of this embodiment, ammonium ferric citrate maybe used at a concentration of, e.g., about 0.01 mg/mL to about 0.05mg/mL about 0.01 mg/mL to about 0.1 mg/mL, about 0.01 mg/mL to about 0.5mg/mL, about 0.05 mg/mL to about 0.1 mg/mL, about 0.05 mg/mL to about0.5 mg/mL, about 0.05 mg/mL to about 1.0 mg/mL, about 0.05 mg/mL toabout 5.0 mg/mL, about 0.1 mg/mL to about 0.5 mg/mL, about 0.1 mg/mL toabout 1.0 mg/mL, about 0.1 mg/mL to about 5.0 mg/mL, about 0.1 mg/mL toabout 10 mg/mL, about 0.1 mg/mL to about 15 mg/mL, about 0.5 mg/mL toabout 1.0 mg/mL, about 0.5 mg/mL to about 5.0 mg/mL, about 0.5 mg/mL toabout 10 mg/mL, about 0.5 mg/mL to about 15 mg/mL, about 1.0 mg/mL toabout 5.0 mg/mL, about 1.0 mg/mL to about 10 mg/mL, about 1.0 mg/mL toabout 15 mg/mL, about 5.0 mg/mL to about 10 mg/mL, about 5.0 mg/mL toabout 15 mg/mL, or about 10 mg/mL to about 15 mg/mL.

An enrichment media typically comprises a growth enhancing agent asdisclosed herein in the concentration ranges disclosed herein.

In aspects of this embodiment, an enrichment media comprises 4.5 g/L SoyPeptone, 7.2 g/L Sodium Chloride, 1.26 g/L Potassium Phosphate(monobasic), 0.18 g/L Potassium Phosphate (dibasic), 13.58 g/L MagnesiumChloride (anhydrous), 0.036 g/L Malachite Green, and 0.62 g/L AmmoniumFerric Citrate. In aspects of this embodiment, an enrichment mediacomprises 4.5 g/L Soy Peptone, 7.2 g/L Sodium Chloride, 1.26 g/LPotassium Phosphate (monobasic), 0.18 g/L Potassium Phosphate (dibasic),29.0 g/L Magnesium Chloride (hexahydrate), 0.036 g/L Malachite Green,and 0.62 g/L Ammonium Ferric Citrate.

Aspect of the present specification disclose, in part, incubation of analiquiot of pre-enrichment media in an enrichment media. Incubation ofan aliquiot of pre-enrichment media is performed under temperature andtime parameters that facilitate the growth of the pathogen in thesample, retard the growth of an unwanted organism in the sample, and/orestablish conditions that otherwise increase the population of thepathogen in the sample and/or retard the growth of an unwanted organismin the sample. Incubation of enrichment media may be performed underconstant rotation, reversal or agitation.

Any temperature may be used during incubation of an aliquiot ofpre-enrichment media in an enrichment media, with the proviso that thetemperature is useful to practice the methods disclosed herein. Inaspects of this embodiment, a temperature used to incubate an aliquiotof pre-enrichment media in an enrichment media may be, e.g., about 15°C., about 16° C., about 17° C., about 18° C., about 19° C., about 20°C., about 21° C., about 22° C., about 23° C., about 24° C., about 25°C., about 26° C., about 27° C., about 28° C., about 29° C., about 30°C., about 31° C., about 32° C., about 33° C., about 34° C., about 35°C., about 36° C., about 37° C., about 38° C., about 39° C., about 40°C., about 41° C., about 42° C., about 43° C., about 44° C., about 45°C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50°C. In other aspects of this embodiment, a temperature used to incubatean aliquiot of pre-enrichment media in an enrichment media may be, e.g.,at least 15° C., at least 16° C., at least 17° C., at least 18° C., atleast 19° C., at least 20° C., at least 21° C., at least 22° C., atleast 23° C., at least 24° C., at least 25° C., at least 26° C., atleast 27° C., at least 28° C., at least 29° C., at least 30° C., atleast 31° C., at least 32° C., at least 33° C., at least 34° C., atleast 35° C., at least 36° C., at least 37° C., at least 38° C., atleast 39° C., at least 40° C., at least 41° C., at least 42° C., atleast 43° C., at least 44° C., at least 45° C., at least 46° C., atleast 47° C., at least 48° C., at least 49° C., or at least 50° C. Inyet other aspects of this embodiment, a temperature used to incubate analiquiot of pre-enrichment media in an enrichment media may be, e.g., atmost 15° C., at most 16° C., at most 17° C., at most 18° C., at most 19°C., at most 20° C., at most 21° C., at most 22° C., at most 23° C., atmost 24° C., at most 25° C., at most 26° C., at most 27° C., at most 28°C., at most 29° C., at most 30° C., at most 31° C., at most 32° C., atmost 33° C., at most 34° C., at most 35° C., at most 36° C., at most 37°C., at most 38° C., at most 39° C., at most 40° C., at most 41° C., atmost 42° C., at most 43° C., at most 44° C., at most 45° C., at most 46°C., at most 47° C., at most 48° C., at most 49° C., or at most 50° C. Instill other aspects of this embodiment, a temperature used to incubatean aliquiot of pre-enrichment media in an enrichment media may be, e.g.,about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C.to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23°C., about 20° C. to about 24° C., about 21° C. to about 25° C., about22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. toabout 28° C., about 25° C. to about 29° C., about 26° C. to about 30°C., about 27° C. to about 31° C., about 28° C. to about 32° C., about29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. toabout 35° C., about 32° C. to about 36° C., about 33° C. to about 37°C., about 34° C. to about 38° C., about 35° C. to about 39° C., about36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. toabout 42° C., about 39° C. to about 43° C., about 40° C. to about 44°C., about 41° C. to about 45° C., about 42° C. to about 46° C., about43° C. to about 47° C., about 44° C. to about 48° C., about 45° C. toabout 49° C., about 46° C. to about 50° C., about 47° C. to about 51°C., about 48° C. to about 52° C., about 49° C. to about 53° C., or about50° C. to about 54° C.

Any time may be used during incubation of an aliquiot of pre-enrichmentmedia in an enrichment media, with the proviso that the time is usefulto practice the methods disclosed herein. In aspects of this embodiment,a time used to incubate an aliquiot of pre-enrichment media in anenrichment media may be, e.g., about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours,about 23 hours, or about 24 hours. In other aspects of this embodiment,a time used to incubate an aliquiot of pre-enrichment media in anenrichment media may be, e.g., at least 2 hours, at least 3 hours, atleast 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, atleast 8 hours, at least 9 hours, at least 10 hours, at least 11 hours,at least 12 hours, at least 13 hours, at least 14 hours, at least 15hours, at least 16 hours, at least 17 hours, at least 18 hours, at least19 hours, at least 20 hours, at least 21 hours, at least 22 hours, atleast 23 hours, or at least 24 hours. In yet other aspects of thisembodiment, a time used to incubate an aliquiot of pre-enrichment mediain an enrichment media may be, e.g., at most 2 hours, at most 3 hours,at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, atmost 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, atmost 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, atmost 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, atmost 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, orat most 24 hours. In yet other aspects of this embodiment, a time usedto incubate an aliquiot of pre-enrichment media in an enrichment mediamay be, e.g., about 2 hours to about 4 hours, about 3 hours to about 5hours, about 4 hours to about 6 hours, about 5 hours to about 7 hours,about 6 hours to about 8 hours, about 7 hours to about 9 hours, about 8hours to about 10 hours, about 9 hours to about 11 hours, about 10 hoursto about 12 hours, about 11 hours to about 13 hours, about 12 hours toabout 14 hours, about 13 hours to about 15 hours, about 14 hours toabout 16 hours, about 15 hours to about 17 hours, about 16 hours toabout 18 hours, about 17 hours to about 19 hours, about 18 hours toabout 20 hours, about 19 hours to about 21 hours, about 20 hours toabout 22 hours, about 21 hours to about 23 hours, about 22 hours toabout 24 hours, about 23 hours to about 25 hours, or about 24 hours toabout 26 hours.

In aspects of this embodiment, an aliquiot of pre-enrichment media in anenrichment media may be incubated at a temperature of, e.g., about 15°C., about 16° C., about 17° C., about 18° C., about 19° C., about 20°C., about 21° C., about 22° C., about 23° C., about 24° C., about 25°C., about 26° C., about 27° C., about 28° C., about 29° C., about 30°C., about 31° C., about 32° C., about 33° C., about 34° C., about 35°C., about 36° C., about 37° C., about 38° C., about 39° C., about 40°C., about 41° C., about 42° C., about 43° C., about 44° C., about 45°C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50°C. for a time of, e.g., about 2 hours, about 3 hours, about 4 hours,about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours,about 23 hours, or about 24 hours. Incubation of the enrichment mediamay be under constant rotation, reversal or agitation.

In other aspects of this embodiment, an aliquiot of pre-enrichment mediain an enrichment media may be incubated at a temperature of, e.g., atleast 15° C., at least 16° C., at least 17° C., at least 18° C., atleast 19° C., at least 20° C., at least 21° C., at least 22° C., atleast 23° C., at least 24° C., at least 25° C., at least 26° C., atleast 27° C., at least 28° C., at least 29° C., at least 30° C., atleast 31° C., at least 32° C., at least 33° C., at least 34° C., atleast 35° C., at least 36° C., at least 37° C., at least 38° C., atleast 39° C., at least 40° C., at least 41° C., at least 42° C., atleast 43° C., at least 44° C., at least 45° C., at least 46° C., atleast 47° C., at least 48° C., at least 49° C., or at least 50° C. for atime of, e.g., at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, atleast 9 hours, at least 10 hours, at least 11 hours, at least 12 hours,at least 13 hours, at least 14 hours, at least 15 hours, at least 16hours, at least 17 hours, at least 18 hours, at least 19 hours, at least20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or atleast 24 hours. Incubation of the enrichment media may be under constantrotation, reversal or agitation.

In yet other aspects of this embodiment, an aliquiot of pre-enrichmentmedia in an enrichment media may be incubated at a temperature of, e.g.,at most 15° C., at most 16° C., at most 17° C., at most 18° C., at most19° C., at most 20° C., at most 21° C., at most 22° C., at most 23° C.,at most 24° C., at most 25° C., at most 26° C., at most 27° C., at most28° C., at most 29° C., at most 30° C., at most 31° C., at most 32° C.,at most 33° C., at most 34° C., at most 35° C., at most 36° C., at most37° C., at most 38° C., at most 39° C., at most 40° C., at most 41° C.,at most 42° C., at most 43° C., at most 44° C., at most 45° C., at most46° C., at most 47° C., at most 48° C., at most 49° C., or at most 50°C. for a time of, e.g., at most 2 hours, at most 3 hours, at most 4hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20hours, at most 21 hours, at most 22 hours, at most 23 hours, or at most24 hours. Incubation of the enrichment media may be under constantrotation, reversal or agitation.

In still other aspects of this embodiment, an aliquiot of pre-enrichmentmedia in an enrichment media may be incubated at a temperature of, e.g.,about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C.to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23°C., about 20° C. to about 24° C., about 21° C. to about 25° C., about22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. toabout 28° C., about 25° C. to about 29° C., about 26° C. to about 30°C., about 27° C. to about 31° C., about 28° C. to about 32° C., about29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. toabout 35° C., about 32° C. to about 36° C., about 33° C. to about 37°C., about 34° C. to about 38° C., about 35° C. to about 39° C., about36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. toabout 42° C., about 39° C. to about 43° C., about 40° C. to about 44°C., about 41° C. to about 45° C., about 42° C. to about 46° C., about43° C. to about 47° C., about 44° C. to about 48° C., about 45° C. toabout 49° C., about 46° C. to about 50° C., about 47° C. to about 51°C., about 48° C. to about 52° C., about 49° C. to about 53° C., or about50° C. to about 54° C. for a time of, e.g., about 2 hours to about 4hours, about 3 hours to about 5 hours, about 4 hours to about 6 hours,about 5 hours to about 7 hours, about 6 hours to about 8 hours, about 7hours to about 9 hours, about 8 hours to about 10 hours, about 9 hoursto about 11 hours, about 10 hours to about 12 hours, about 11 hours toabout 13 hours, about 12 hours to about 14 hours, about 13 hours toabout 15 hours, about 14 hours to about 16 hours, about 15 hours toabout 17 hours, about 16 hours to about 18 hours, about 17 hours toabout 19 hours, about 18 hours to about 20 hours, about 19 hours toabout 21 hours, about 20 hours to about 22 hours, about 21 hours toabout 23 hours, about 22 hours to about 24 hours, about 23 hours toabout 25 hours, or about 24 hours to about 26 hours. Incubation of theenrichment media may be under constant rotation, reversal or agitation.

Aspect of the present specification disclose, in part, a purificationstep. After being enriched, a pathogen may be subsequently purified froman enrichment media. Purification of a pathogen includes capture of thepathogen to a more concentrated form and/or removal contaminatingmicroorganism, impurities, and debris. As such a purification stepdisclosed herein increases detection of a pathogen by increasing theconcentration of pathogen and/or decreasing contaminants, therebyincreasing the level of detectable signal measured in a subsequentdetection step. Common purification procedures used to capture apathogen and/or remove contaminating microorganism, impurities, anddebris include affinity chromatography, ion-exchange chromatography,size exclusion chromatography, hydrophobic-interaction chromatography,ceramic hydroxyapatite chromatography, reverse-phase HPLC, gelfiltration, precipitation, immuno-precipitation, diafiltration,chromatofocusing.

In one embodiment, a pathogen is purified from an enrichment media usingan immuno-precipitation using antibodies or aptamers for a pathogen ofinterest. Immunoprecipitation is the technique of precipitating anantigen out of solution using an antibody or aptamer that specificallybinds to that antigen. Immunoprecipitation requires that the antibody becoupled to a solid substrate at some point in the procedure. This istypically done using standard coupling procedures known in the art.Examples of solid substrates include agarose particles and magneticparticles. In aspects of this embodiment, the immuno-precipitationmethod employs antibodies or aptamers for a pathogen linked to magneticparticles.

Any concentration of magnetic particles linked with an antibody orapatmer may be used during its incubation during a purification step,with the proviso that the concentration is useful to practice themethods disclosed herein. In aspects of this embodiment, a concentrationof magnetic particles linked with an antibody or apatmer used during apurification step may be, e.g., about 1×10⁴ immunmagnetic particles/mLof enrichment media comprising the pathogen of interest, about 1×10⁵immunmagnetic particles/mL of enrichment media comprising the pathogenof interest, about 1×10⁶ immunmagnetic particles/mL of enrichment mediacomprising the pathogen of interest, about 1×10⁷ immunmagneticparticles/mL of enrichment media comprising the pathogen of interest, orabout 1×10⁸ immunmagnetic particles/mL of enrichment media comprisingthe pathogen of interest. In other aspects of this embodiment, aconcentration of magnetic particles linked with an antibody or apatmerused during a purification step may be, e.g., at least 1×10⁴immunmagnetic particles/mL of enrichment media comprising the pathogenof interest, at least 1×10⁵ immunmagnetic particles/mL of enrichmentmedia comprising the pathogen of interest, at least 1×10⁶ immunmagneticparticles/mL of enrichment media comprising the pathogen of interest, atleast 1×10⁷ immunmagnetic particles/mL of enrichment media comprisingthe pathogen of interest, or at least 1×10⁸ immunmagnetic particles/mLof enrichment media comprising the pathogen of interest. In yet otheraspects of this embodiment, a concentration of magnetic particles linkedwith an antibody or apatmer used during a purification step may be,e.g., at most 1×10⁴ immunmagnetic particles/mL of enrichment mediacomprising the pathogen of interest, at most 1×10⁵ immunmagneticparticles/mL of enrichment media comprising the pathogen of interest, atmost 1×10⁶ immunmagnetic particles/mL of enrichment media comprising thepathogen of interest, at most 1×10⁷ immunmagnetic particles/mL ofenrichment media comprising the pathogen of interest, or at most 1×10⁸immunmagnetic particles/mL of enrichment media comprising the pathogenof interest.

In still other aspects of this embodiment, a concentration of magneticparticles linked with an antibody or apatmer used during a purificationstep may be, e.g., about 1×10⁴ to about 1×10⁵ immunmagnetic particles/mLof enrichment media comprising the pathogen of interest, about 1×10⁴ toabout 1×10⁶ immunmagnetic particles/mL of enrichment media comprisingthe pathogen of interest, about 1×10⁴ to about 1×10⁷ immunmagneticparticles/mL of enrichment media comprising the pathogen of interest,about 1×10⁴ to about 1×10⁸ immunmagnetic particles/mL of enrichmentmedia comprising the pathogen of interest, about 1×10⁵ to about 1×10⁶immunmagnetic particles/mL of enrichment media comprising the pathogenof interest, about 1×10⁵ to about 1×10⁷ immunmagnetic particles/mL ofenrichment media comprising the pathogen of interest, about 1×10⁵ toabout 1×10⁸ immunmagnetic particles/mL of enrichment media comprisingthe pathogen of interest, about 1×10⁶ to about 1×10⁷ immunmagneticparticles/mL of enrichment media comprising the pathogen of interest,about 1×10⁶ to about 1×10⁸ immunmagnetic particles/mL of enrichmentmedia comprising the pathogen of interest, or about 1×10⁷ to about 1×10⁸immunmagnetic particles/mL of enrichment media comprising the pathogenof interest.

Aspect of the present specification disclose, in part, incubation ofimmunmagnetic particles with enrichment media comprising the pathogen ofinterest. Incubation of immunmagnetic particles with enrichment mediacomprising the pathogen of interest is performed under temperature andtime parameters that facilitate binding of the pathogen to the magneticparticles linked with an antibody or apatmer for the pathogen ofinterest. Incubation of immunmagnetic particles with enrichment mediacomprising the pathogen of interest may be performed underagitation/rotation.

Any temperature may be used during incubation of immunmagnetic particleswith enrichment media comprising the pathogen of interest, with theproviso that the temperature is useful to practice the methods disclosedherein. In aspects of this embodiment, a temperature used to incubateimmunmagnetic particles with enrichment media comprising the pathogen ofinterest may be, e.g., about 15° C., about 16° C., about 17° C., about18° C., about 19° C., about 20° C., about 21° C., about 22° C., about23° C., about 24° C., about 25° C., about 26° C., about 27° C., about28° C., about 29° C., about 30° C., about 31° C., about 32° C., about33° C., about 34° C., about 35° C., about 36° C., about 37° C., about38° C., about 39° C., about 40° C., about 41° C., about 42° C., about43° C., about 44° C., about 45° C., about 46° C., about 47° C., about48° C., about 49° C., or about 50° C. In other aspects of thisembodiment, a temperature used to incubate immunmagnetic particles withenrichment media comprising the pathogen of interest may be, e.g., atleast 15° C., at least 16° C., at least 17° C., at least 18° C., atleast 19° C., at least 20° C., at least 21° C., at least 22° C., atleast 23° C., at least 24° C., at least 25° C., at least 26° C., atleast 27° C., at least 28° C., at least 29° C., at least 30° C., atleast 31° C., at least 32° C., at least 33° C., at least 34° C., atleast 35° C., at least 36° C., at least 37° C., at least 38° C., atleast 39° C., at least 40° C., at least 41° C., at least 42° C., atleast 43° C., at least 44° C., at least 45° C., at least 46° C., atleast 47° C., at least 48° C., at least 49° C., or at least 50° C. Inyet other aspects of this embodiment, a temperature used to incubateimmunmagnetic particles with enrichment media comprising the pathogen ofinterest may be, e.g., at most 15° C., at most 16° C., at most 17° C.,at most 18° C., at most 19° C., at most 20° C., at most 21° C., at most22° C., at most 23° C., at most 24° C., at most 25° C., at most 26° C.,at most 27° C., at most 28° C., at most 29° C., at most 30° C., at most31° C., at most 32° C., at most 33° C., at most 34° C., at most 35° C.,at most 36° C., at most 37° C., at most 38° C., at most 39° C., at most40° C., at most 41° C., at most 42° C., at most 43° C., at most 44° C.,at most 45° C., at most 46° C., at most 47° C., at most 48° C., at most49° C., or at most 50° C. In still other aspects of this embodiment, atemperature used to incubate immunmagnetic particles with enrichmentmedia comprising the pathogen of interest may be, e.g., about 15° C. toabout 19° C., about 16° C. to about 20° C., about 17° C. to about 21°C., about 18° C. to about 22° C., about 19° C. to about 23° C., about20° C. to about 24° C., about 21° C. to about 25° C., about 22° C. toabout 26° C., about 23° C. to about 27° C., about 24° C. to about 28°C., about 25° C. to about 29° C., about 26° C. to about 30° C., about27° C. to about 31° C., about 28° C. to about 32° C., about 29° C. toabout 33° C., about 30° C. to about 34° C., about 31° C. to about 35°C., about 32° C. to about 36° C., about 33° C. to about 37° C., about34° C. to about 38° C., about 35° C. to about 39° C., about 36° C. toabout 40° C., about 37° C. to about 41° C., about 38° C. to about 42°C., about 39° C. to about 43° C., about 40° C. to about 44° C., about41° C. to about 45° C., about 42° C. to about 46° C., about 43° C. toabout 47° C., about 44° C. to about 48° C., about 45° C. to about 49°C., about 46° C. to about 50° C., about 47° C. to about 51° C., about48° C. to about 52° C., about 49° C. to about 53° C., or about 50° C. toabout 54° C.

Any time may be used during incubation of immunmagnetic particles withenrichment media comprising the pathogen of interest, with the provisothat the time is useful to practice the methods disclosed herein. Inaspects of this embodiment, a time used to incubate immunmagneticparticles with enrichment media comprising the pathogen of interest maybe, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100minutes, about 110 minutes, about 120 minutes, about 130 minutes, about140 minutes, or about 150 minutes. In other aspects of this embodiment,a time used to incubate immunmagnetic particles with enrichment mediacomprising the pathogen of interest may be, e.g., at least 5 minutes, atleast 10 minutes, at least 15 minutes, at least 20 minutes, at least 30minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes,at least 70 minutes, at least 80 minutes, at least 90 minutes, at least100 minutes, at least 110 minutes, at least 120 minutes, at least 130minutes, at least 140 minutes, or at least 150 minutes. In yet otheraspects of this embodiment, a time used to incubate immunmagneticparticles with enrichment media comprising the pathogen of interest maybe, e.g., at most 5 minutes, at most 10 minutes, at most 15 minutes, atmost 20 minutes, at most 30 minutes, at most 40 minutes, at most 50minutes, at most 60 minutes, at most 70 minutes, at most 80 minutes, atmost 90 minutes, at most 100 minutes, at most 110 minutes, at most 120minutes, at most 130 minutes, at most 140 minutes, or at most 150minutes.

In yet other aspects of this embodiment, a time used to incubateimmunmagnetic particles with enrichment media comprising the pathogen ofinterest may be, e.g., about 5 minutes to about 20 minutes, about 5minutes to about 30 minutes, about 5 minutes to about 40 minutes, about5 minutes to about 50 minutes, about 5 minutes to about 60 minutes,about 5 minutes to about 70 minutes, about 5 minutes to about 80minutes, about 5 minutes to about 90 minutes, about 5 minutes to about100 minutes, about 5 minutes to about 110 minutes, about 5 minutes toabout 120 minutes, about 5 minutes to about 130 minutes, about 5 minutesto about 140 minutes, about 5 minutes to about 150 minutes, about 10minutes to about 20 minutes, about 10 minutes to about 30 minutes, about10 minutes to about 40 minutes, about 10 minutes to about 50 minutes,about 10 minutes to about 60 minutes, about 10 minutes to about 70minutes, about 10 minutes to about 80 minutes, about 10 minutes to about90 minutes, about 10 minutes to about 100 minutes, about 10 minutes toabout 110 minutes, about 10 minutes to about 120 minutes, about 10minutes to about 130 minutes, about 10 minutes to about 140 minutes,about 10 minutes to about 150 minutes, about 20 minutes to about 30minutes, about 20 minutes to about 40 minutes, about 20 minutes to about50 minutes, about 20 minutes to about 60 minutes, about 20 minutes toabout 70 minutes, about 20 minutes to about 80 minutes, about 20 minutesto about 90 minutes, about 20 minutes to about 100 minutes, about 20minutes to about 110 minutes, about 20 minutes to about 120 minutes,about 20 minutes to about 130 minutes, about 20 minutes to about 140minutes, about 20 minutes to about 150 minutes, about 30 minutes toabout 40 minutes, about 30 minutes to about 50 minutes, about 30 minutesto about 60 minutes, about 30 minutes to about 70 minutes, about 30minutes to about 80 minutes, about 30 minutes to about 90 minutes, about30 minutes to about 100 minutes, about 30 minutes to about 110 minutes,about 30 minutes to about 120 minutes, about 30 minutes to about 130minutes, about 30 minutes to about 140 minutes, about 30 minutes toabout 150 minutes, about 60 minutes to about 70 minutes, about 60minutes to about 80 minutes, about 60 minutes to about 90 minutes, about60 minutes to about 100 minutes, about 60 minutes to about 110 minutes,about 60 minutes to about 120 minutes, about 60 minutes to about 130minutes, about 60 minutes to about 140 minutes, about 60 minutes toabout 150 minutes, about 90 minutes to about 100 minutes, about 90minutes to about 110 minutes, about 90 minutes to about 120 minutes,about 90 minutes to about 130 minutes, about 90 minutes to about 140minutes, about 90 minutes to about 150 minutes, about 120 minutes toabout 130 minutes, about 120 minutes to about 140 minutes, or about 120minutes to about 150 minutes.

In aspects of this embodiment, immunmagnetic particles may be incubatedwith enrichment media comprising the pathogen of interest at atemperature of, e.g., about 15° C., about 16° C., about 17° C., about18° C., about 19° C., about 20° C., about 21° C., about 22° C., about23° C., about 24° C., about 25° C., about 26° C., about 27° C., about28° C., about 29° C., about 30° C., about 31° C., about 32° C., about33° C., about 34° C., about 35° C., about 36° C., about 37° C., about38° C., about 39° C., about 40° C., about 41° C., about 42° C., about43° C., about 44° C., about 45° C., about 46° C., about 47° C., about48° C., about 49° C., or about 50° C. for a time of, e.g., about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70minutes, about 80 minutes, about 90 minutes, about 100 minutes, about110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, orabout 150 minutes.

In other aspects of this embodiment, immunmagnetic particles may beincubated with enrichment media comprising the pathogen of interest at atemperature of, e.g., at least 15° C., at least 16° C., at least 17° C.,at least 18° C., at least 19° C., at least 20° C., at least 21° C., atleast 22° C., at least 23° C., at least 24° C., at least 25° C., atleast 26° C., at least 27° C., at least 28° C., at least 29° C., atleast 30° C., at least 31° C., at least 32° C., at least 33° C., atleast 34° C., at least 35° C., at least 36° C., at least 37° C., atleast 38° C., at least 39° C., at least 40° C., at least 41° C., atleast 42° C., at least 43° C., at least 44° C., at least 45° C., atleast 46° C., at least 47° C., at least 48° C., at least 49° C., or atleast 50° C. for a time of, e.g., at least 5 minutes, at least 10minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes,at least 40 minutes, at least 50 minutes, at least 60 minutes, at least70 minutes, at least 80 minutes, at least 90 minutes, at least 100minutes, at least 110 minutes, at least 120 minutes, at least 130minutes, at least 140 minutes, or at least 150 minutes.

In yet other aspects of this embodiment, immunmagnetic particles may beincubated with enrichment media comprising the pathogen of interest at atemperature of, e.g., at most 15° C., at most 16° C., at most 17° C., atmost 18° C., at most 19° C., at most 20° C., at most 21° C., at most 22°C., at most 23° C., at most 24° C., at most 25° C., at most 26° C., atmost 27° C., at most 28° C., at most 29° C., at most 30° C., at most 31°C., at most 32° C., at most 33° C., at most 34° C., at most 35° C., atmost 36° C., at most 37° C., at most 38° C., at most 39° C., at most 40°C., at most 41° C., at most 42° C., at most 43° C., at most 44° C., atmost 45° C., at most 46° C., at most 47° C., at most 48° C., at most 49°C., or at most 50° C. for a time of, e.g., at most 5 minutes, at most 10minutes, at most 15 minutes, at most 20 minutes, at most 30 minutes, atmost 40 minutes, at most 50 minutes, at most 60 minutes, at most 70minutes, at most 80 minutes, at most 90 minutes, at most 100 minutes, atmost 110 minutes, at most 120 minutes, at most 130 minutes, at most 140minutes, or at most 150 minutes.

In still other aspects of this embodiment, immunmagnetic particles maybe incubated with enrichment media comprising the pathogen of interestat a temperature of, e.g., about 15° C. to about 19° C., about 16° C. toabout 20° C., about 17° C. to about 21° C., about 18° C. to about 22°C., about 19° C. to about 23° C., about 20° C. to about 24° C., about21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. toabout 27° C., about 24° C. to about 28° C., about 25° C. to about 29°C., about 26° C. to about 30° C., about 27° C. to about 31° C., about28° C. to about 32° C., about 29° C. to about 33° C., about 30° C. toabout 34° C., about 31° C. to about 35° C., about 32° C. to about 36°C., about 33° C. to about 37° C., about 34° C. to about 38° C., about35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. toabout 41° C., about 38° C. to about 42° C., about 39° C. to about 43°C., about 40° C. to about 44° C., about 41° C. to about 45° C., about42° C. to about 46° C., about 43° C. to about 47° C., about 44° C. toabout 48° C., about 45° C. to about 49° C., about 46° C. to about 50°C., about 47° C. to about 51° C., about 48° C. to about 52° C., about49° C. to about 53° C., or about 50° C. to about 54° C. for a time of,e.g., about 5 minutes to about 20 minutes, about 5 minutes to about 30minutes, about 5 minutes to about 40 minutes, about 5 minutes to about50 minutes, about 5 minutes to about 60 minutes, about 5 minutes toabout 70 minutes, about 5 minutes to about 80 minutes, about 5 minutesto about 90 minutes, about 5 minutes to about 100 minutes, about 5minutes to about 110 minutes, about 5 minutes to about 120 minutes,about 5 minutes to about 130 minutes, about 5 minutes to about 140minutes, about 5 minutes to about 150 minutes, about 10 minutes to about20 minutes, about 10 minutes to about 30 minutes, about 10 minutes toabout 40 minutes, about 10 minutes to about 50 minutes, about 10 minutesto about 60 minutes, about 10 minutes to about 70 minutes, about 10minutes to about 80 minutes, about 10 minutes to about 90 minutes, about10 minutes to about 100 minutes, about 10 minutes to about 110 minutes,about 10 minutes to about 120 minutes, about 10 minutes to about 130minutes, about 10 minutes to about 140 minutes, about 10 minutes toabout 150 minutes, about 20 minutes to about 30 minutes, about 20minutes to about 40 minutes, about 20 minutes to about 50 minutes, about20 minutes to about 60 minutes, about 20 minutes to about 70 minutes,about 20 minutes to about 80 minutes, about 20 minutes to about 90minutes, about 20 minutes to about 100 minutes, about 20 minutes toabout 110 minutes, about 20 minutes to about 120 minutes, about 20minutes to about 130 minutes, about 20 minutes to about 140 minutes,about 20 minutes to about 150 minutes, about 30 minutes to about 40minutes, about 30 minutes to about 50 minutes, about 30 minutes to about60 minutes, about 30 minutes to about 70 minutes, about 30 minutes toabout 80 minutes, about 30 minutes to about 90 minutes, about 30 minutesto about 100 minutes, about 30 minutes to about 110 minutes, about 30minutes to about 120 minutes, about 30 minutes to about 130 minutes,about 30 minutes to about 140 minutes, about 30 minutes to about 150minutes, about 60 minutes to about 70 minutes, about 60 minutes to about80 minutes, about 60 minutes to about 90 minutes, about 60 minutes toabout 100 minutes, about 60 minutes to about 110 minutes, about 60minutes to about 120 minutes, about 60 minutes to about 130 minutes,about 60 minutes to about 140 minutes, about 60 minutes to about 150minutes, about 90 minutes to about 100 minutes, about 90 minutes toabout 110 minutes, about 90 minutes to about 120 minutes, about 90minutes to about 130 minutes, about 90 minutes to about 140 minutes,about 90 minutes to about 150 minutes, about 120 minutes to about 130minutes, about 120 minutes to about 140 minutes, or about 120 minutes toabout 150 minutes.

After completion of incubation, the pathogen-bound immunoparticles maybe isolated from the enrichment media. In aspects of this embodiment,immunoparticles may be isolated from the enrichment media using amagnetic separator which concentrates the immunoparticles in a specificlocation allowing for the enrichment media to be removed. Alternatively,immunoparticles may be isolated from the enrichment media by centrifugedto concentrate the immunoparticles, thereby allowing for the enrichmentmedia to be removed. After removal of the enrichment media, the isolatedpathogen-bound immunoparticles may be washed one or more times using abuffered solution. Subsequent isolation of the immunoparticles may beaccomplished using a magnetic separator or centrifugation and removal ofthe buffered solution.

However, a purification step disclosed herein is optional. Thus, in oneembodiment, a method of detecting a pathogen disclosed herein does notcomprise a purification of a pathogen from a pre-enrichment media, anenrichment media, or both a pre-enrichment media and an enrichmentmedia.

Aspect of the present specification disclose, in part, a detection step.After being enriched, presence or absence of a pathogen of interest maybe determined by qualitatively or quantitatively measuring the amount ofpathogen contained within the enrichment media. In one embodiment,detection presence or absence of a pathogen of interest occurs aftercompletion of an enrichment step disclosed herein and without the needof a purification step disclosed herein. In one embodiment, detectionpresence or absence of a pathogen of interest occurs after completion ofa purification step disclosed herein. However, an enrichment mediacomprising a pathogen of interest may be processed to remove debris andother contaminants without any pathogen purification. In one embodiment,an enrichment media comprising a pathogen of interest is centrifuged toremove debris. In another embodiment no such centrifugation step isrequired. In addition, use of a secondary antibody to boast a detectionsignal is optional. Thus, in one embodiment, a method of detecting apathogen does not comprise use of a secondary antibody to boast adetection signal.

In aspects of this embodiment, a method disclosed herein mayqualitatively or quantitatively detect pathogen having a concentrationin a sample disclosed herein of, e.g., about 1×10⁻⁵ cfu/mL, about 1×10⁻⁴cfu/mL, about 1×10⁻³ cfu/mL, about 1×10⁻² cfu/mL, about 1×10⁻¹ cfu/mL,about 1×10⁰ cfu/mL, about 1×10¹ cfu/mL, about 1×10² cfu/mL, about 1×10³cfu/mL, about 1×10⁴ cfu/mL, about 1×10⁵ cfu/mL, about 1×10⁶ cfu/mL,about 1×10⁷ cfu/mL, about 1×10⁸ cfu/mL, about 1×10⁹ cfu/mL, or about1×10¹⁰ cfu/mL. In other aspects of this embodiment, a method disclosedherein may qualitatively or quantitatively detect pathogen having aconcentration in a sample disclosed herein of, e.g., at least 1×10⁻⁵cfu/mL, at least 1×10⁻⁴ cfu/mL, at least 1×10⁻³ cfu/mL, at least 1×10⁻²cfu/mL, at least 1×10⁻¹ cfu/mL, at least 1×10⁰ cfu/mL, at least 1×10¹cfu/mL, at least 1×10² cfu/mL, at least 1×10³ cfu/mL, at least 1×10⁴cfu/mL, at least 1×10⁵ cfu/mL, at least 1×10⁶ cfu/mL, at least 1×10⁷cfu/mL, at least 1×10⁸ cfu/mL, at least 1×10⁹ cfu/mL, or at least 1×10¹⁰cfu/mL. In yet other aspects of this embodiment, a method disclosedherein may qualitatively or quantitatively detect pathogen having aconcentration in a sample disclosed herein of, e.g., at most 1×10⁻⁵cfu/mL, at most 1×10⁻⁴ cfu/mL, at most 1×10⁻³ cfu/mL, at most 1×10⁻²cfu/mL, at most 1×10⁻¹ cfu/mL, at most 1×10⁰ cfu/mL, at most 1×10¹cfu/mL, at most 1×10² cfu/mL, at most 1×10³ cfu/mL, at most 1×10⁴cfu/mL, at most 1×10⁵ cfu/mL, at most 1×10⁶ cfu/mL, at most 1×10⁷cfu/mL, at most 1×10⁸ cfu/mL, at most 1×10⁹ cfu/mL, or at most 1×10¹⁰cfu/mL.

In still other aspects of this embodiment, a method disclosed herein mayqualitatively or quantitatively detect pathogen having a concentrationin a sample disclosed herein of, e.g., about 1×10⁻⁵ cfu/mL to about1×10⁻⁴ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁻³ cfu/mL, about 1×10⁻⁵cfu/mL to about 1×10⁻² cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁻¹cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁰ cfu/mL, about 1×10⁻⁵ cfu/mLto about 1×10¹ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10² cfu/mL, about1×10⁻⁵ cfu/mL to about 1×10³ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁴cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁵ cfu/mL, about 1×10⁻⁵ cfu/mLto about 1×10⁶ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁷ cfu/mL, about1×10⁻⁵ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁹cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10¹⁰ cfu/mL, about 1×10⁻⁴ cfu/mLto about 1×10⁻³ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁻² cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10⁰ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻⁴cfu/mL to about 1×10² cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10³ cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10⁵ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻⁴cfu/mL to about 1×10⁷ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁸ cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10¹⁰ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁻² cfu/mL, about 1×10⁻³cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁰cfu/mL, about 1×10⁻³ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻³ cfu/mLto about 1×10² cfu/mL, about 1×10⁻³ cfu/mL to about 1×10³ cfu/mL, about1×10⁻³ cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁵cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻³ cfu/mLto about 1×10⁷ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁸ cfu/mL, about1×10⁻³ cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10¹⁰cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻² cfu/mLto about 1×10⁰ cfu/mL, about 1×10⁻² cfu/mL to about 1×10¹ cfu/mL, about1×10⁻² cfu/mL to about 1×10² cfu/mL, about 1×10⁻² cfu/mL to about 1×10³cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻² cfu/mLto about 1×10⁵ cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁶ cfu/mL, about1×10⁻² cfu/mL to about 1×10⁷ cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁸cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻² cfu/mLto about 1×10¹⁰ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁰ cfu/mL, about1×10⁻¹ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10²cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10³ cfu/mL, about 1×10⁻¹ cfu/mLto about 1×10⁴ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁻¹ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁻¹ cfu/mLto about 1×10⁹ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10¹⁰ cfu/mL, about1×10⁰ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁰ cfu/mL to about 1×10²cfu/mL, about 1×10⁰ cfu/mL to about 1×10³ cfu/mL, about 1×10⁰ cfu/mL toabout 1×10⁴ cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁰ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁰ cfu/mL toabout 1×10⁹ cfu/mL, about 1×10⁰ cfu/mL to about 1×10¹⁰ cfu/mL, about1×10¹ cfu/mL to about 1×10² cfu/mL, about 1×10¹ cfu/mL to about 1×10³cfu/mL, about 1×10¹ cfu/mL to about 1×10⁴ cfu/mL, about 1×10¹ cfu/mL toabout 1×10⁵ cfu/mL, about 1×10¹ cfu/mL to about 1×10⁶ cfu/mL, about1×10¹ cfu/mL to about 1×10⁷ cfu/mL, about 1×10¹ cfu/mL to about 1×10⁸cfu/mL, about 1×10¹ cfu/mL to about 1×10⁹ cfu/mL, about 1×10¹ cfu/mL toabout 1×10¹⁰ cfu/mL, about 1×10² cfu/mL to about 1×10³ cfu/mL, about1×10² cfu/mL to about 1×10⁴ cfu/mL, about 1×10² cfu/mL to about 1×10⁵cfu/mL, about 1×10² cfu/mL to about 1×10⁶ cfu/mL, about 1×10² cfu/mL toabout 1×10⁷ cfu/mL, about 1×10² cfu/mL to about 1×10⁸ cfu/mL, about1×10² cfu/mL to about 1×10⁹ cfu/mL, about 1×10² cfu/mL to about 1×10¹⁰cfu/mL, about 1×10³ cfu/mL to about 1×10⁴ cfu/mL, about 1×10³ cfu/mL toabout 1×10⁵ cfu/mL, about 1×10³ cfu/mL to about 1×10⁶ cfu/mL, about1×10³ cfu/mL to about 1×10⁷ cfu/mL, about 1×10³ cfu/mL to about 1×10⁸cfu/mL, about 1×10³ cfu/mL to about 1×10⁹ cfu/mL, about 1×10³ cfu/mL toabout 1×10¹⁰ cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁴ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁴ cfu/mL toabout 1×10⁹ cfu/mL, or about 1×10⁴ cfu/mL to about 1×10¹⁰ cfu/mL.

In aspects of this embodiment, a method disclosed herein mayqualitatively or quantitatively detect pathogen having a concentrationafter the pre-enrichment step of, e.g., about 1×10⁻⁵ cfu/mL, about1×10⁻⁴ cfu/mL, about 1×10⁻³ cfu/mL, about 1×10⁻² cfu/mL, about 1×10⁻¹cfu/mL, about 1×10⁰ cfu/mL, about 1×10¹ cfu/mL, about 1×10² cfu/mL,about 1×10³ cfu/mL, about 1×10⁴ cfu/mL, about 1×10⁵ cfu/mL, about 1×10⁶cfu/mL, about 1×10⁷ cfu/mL, about 1×10⁸ cfu/mL, about 1×10⁹ cfu/mL, orabout 1×10¹⁰ cfu/mL. In other aspects of this embodiment, a methoddisclosed herein may qualitatively or quantitatively detect pathogenhaving a concentration after the pre-enrichment step of, e.g., at least1×10⁻⁵ cfu/mL, at least 1×10⁻⁴ cfu/mL, at least 1×10⁻³ cfu/mL, at least1×10⁻² cfu/mL, at least 1×10⁻¹ cfu/mL, at least 1×10⁰ cfu/mL, at least1×10¹ cfu/mL, at least 1×10² cfu/mL, at least 1×10³ cfu/mL, at least1×10⁴ cfu/mL, at least 1×10⁵ cfu/mL, at least 1×10⁶ cfu/mL, at least1×10⁷ cfu/mL, at least 1×10⁸ cfu/mL, at least 1×10⁹ cfu/mL, or at least1×10¹⁰ cfu/mL. In yet other aspects of this embodiment, a methoddisclosed herein may qualitatively or quantitatively detect pathogenhaving a concentration after the pre-enrichment step of, e.g., at most1×10⁻⁵ cfu/mL, at most 1×10⁻⁴ cfu/mL, at most 1×10⁻³ cfu/mL, at most1×10⁻² cfu/mL, at most 1×10⁻¹ cfu/mL, at most 1×10⁰ cfu/mL, at most1×10¹ cfu/mL, at most 1×10² cfu/mL, at most 1×10³ cfu/mL, at most 1×10⁴cfu/mL, at most 1×10⁵ cfu/mL, at most 1×10⁶ cfu/mL, at most 1×10⁷cfu/mL, at most 1×10⁸ cfu/mL, at most 1×10⁹ cfu/mL, or at most 1×10¹⁰cfu/mL.

In still other aspects of this embodiment, a method disclosed herein mayqualitatively or quantitatively detect pathogen having a concentrationafter the pre-enrichment step off, e.g., about 1×10⁻⁵ cfu/mL to about1×10⁻⁴ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁻³ cfu/mL, about 1×10⁻⁵cfu/mL to about 1×10⁻² cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁻¹cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁰ cfu/mL, about 1×10⁻⁵ cfu/mLto about 1×10¹ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10² cfu/mL, about1×10⁻⁵ cfu/mL to about 1×10³ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁴cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁵ cfu/mL, about 1×10⁻⁵ cfu/mLto about 1×10⁶ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁷ cfu/mL, about1×10⁻⁵ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁹cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10¹⁰ cfu/mL, about 1×10⁻⁴ cfu/mLto about 1×10⁻³ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁻² cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10⁰ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻⁴cfu/mL to about 1×10² cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10³ cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10⁵ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻⁴cfu/mL to about 1×10⁷ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁸ cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10¹⁰ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁻² cfu/mL, about 1×10⁻³cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁰cfu/mL, about 1×10⁻³ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻³ cfu/mLto about 1×10² cfu/mL, about 1×10⁻³ cfu/mL to about 1×10³ cfu/mL, about1×10⁻³ cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁵cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻³ cfu/mLto about 1×10⁷ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁸ cfu/mL, about1×10⁻³ cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10¹⁰cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻² cfu/mLto about 1×10⁰ cfu/mL, about 1×10⁻² cfu/mL to about 1×10¹ cfu/mL, about1×10⁻² cfu/mL to about 1×10² cfu/mL, about 1×10⁻² cfu/mL to about 1×10³cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻² cfu/mLto about 1×10⁵ cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁶ cfu/mL, about1×10⁻² cfu/mL to about 1×10⁷ cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁸cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻² cfu/mLto about 1×10¹⁰ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁰ cfu/mL, about1×10⁻¹ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10²cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10³ cfu/mL, about 1×10⁻¹ cfu/mLto about 1×10⁴ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁻¹ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁻¹ cfu/mLto about 1×10⁹ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10¹⁰ cfu/mL, about1×10⁰ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁰ cfu/mL to about 1×10²cfu/mL, about 1×10⁰ cfu/mL to about 1×10³ cfu/mL, about 1×10⁰ cfu/mL toabout 1×10⁴ cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁰ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁰ cfu/mL toabout 1×10⁹ cfu/mL, about 1×10⁰ cfu/mL to about 1×10¹⁰ cfu/mL, about1×10¹ cfu/mL to about 1×10² cfu/mL, about 1×10¹ cfu/mL to about 1×10³cfu/mL, about 1×10¹ cfu/mL to about 1×10⁴ cfu/mL, about 1×10¹ cfu/mL toabout 1×10⁵ cfu/mL, about 1×10¹ cfu/mL to about 1×10⁶ cfu/mL, about1×10¹ cfu/mL to about 1×10⁷ cfu/mL, about 1×10¹ cfu/mL to about 1×10⁸cfu/mL, about 1×10¹ cfu/mL to about 1×10⁹ cfu/mL, about 1×10¹ cfu/mL toabout 1×10¹⁰ cfu/mL, about 1×10² cfu/mL to about 1×10³ cfu/mL, about1×10² cfu/mL to about 1×10⁴ cfu/mL, about 1×10² cfu/mL to about 1×10⁵cfu/mL, about 1×10² cfu/mL to about 1×10⁶ cfu/mL, about 1×10² cfu/mL toabout 1×10⁷ cfu/mL, about 1×10² cfu/mL to about 1×10⁸ cfu/mL, about1×10² cfu/mL to about 1×10⁹ cfu/mL, about 1×10² cfu/mL to about 1×10¹⁰cfu/mL, about 1×10³ cfu/mL to about 1×10⁴ cfu/mL, about 1×10³ cfu/mL toabout 1×10⁵ cfu/mL, about 1×10³ cfu/mL to about 1×10⁶ cfu/mL, about1×10³ cfu/mL to about 1×10⁷ cfu/mL, about 1×10³ cfu/mL to about 1×10⁸cfu/mL, about 1×10³ cfu/mL to about 1×10⁹ cfu/mL, about 1×10³ cfu/mL toabout 1×10¹⁰ cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁴ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁴ cfu/mL toabout 1×10⁹ cfu/mL, or about 1×10⁴ cfu/mL to about 1×10¹⁰ cfu/mL.

In aspects of this embodiment, a method disclosed herein mayqualitatively or quantitatively detect pathogen having a concentrationafter the enrichment step of, e.g., about 1×10⁻⁵ cfu/mL, about 1×10⁻⁴cfu/mL, about 1×10⁻³ cfu/mL, about 1×10⁻² cfu/mL, about 1×10⁻¹ cfu/mL,about 1×10⁰ cfu/mL, about 1×10¹ cfu/mL, about 1×10² cfu/mL, about 1×10³cfu/mL, about 1×10⁴ cfu/mL, about 1×10⁵ cfu/mL, about 1×10⁶ cfu/mL,about 1×10⁷ cfu/mL, about 1×10⁸ cfu/mL, about 1×10⁹ cfu/mL, or about1×10¹⁰ cfu/mL. In other aspects of this embodiment, a method disclosedherein may qualitatively or quantitatively detect pathogen having aconcentration after the enrichment step of, e.g., at least 1×10⁻⁵cfu/mL, at least 1×10⁻⁴ cfu/mL, at least 1×10⁻³ cfu/mL, at least 1×10⁻²cfu/mL, at least 1×10⁻¹ cfu/mL, at least 1×10⁰ cfu/mL, at least 1×10¹cfu/mL, at least 1×10² cfu/mL, at least 1×10³ cfu/mL, at least 1×10⁴cfu/mL, at least 1×10⁵ cfu/mL, at least 1×10⁶ cfu/mL, at least 1×10⁷cfu/mL, at least 1×10⁸ cfu/mL, at least 1×10⁹ cfu/mL, or at least 1×10¹⁰cfu/mL. In yet other aspects of this embodiment, a method disclosedherein may qualitatively or quantitatively detect pathogen having aconcentration after the enrichment step of, e.g., at most 1×10⁻⁵ cfu/mL,at most 1×10⁻⁴ cfu/mL, at most 1×10⁻³ cfu/mL, at most 1×10⁻² cfu/mL, atmost 1×10⁻¹ cfu/mL, at most 1×10⁰ cfu/mL, at most 1×10¹ cfu/mL, at most1×10² cfu/mL, at most 1×10³ cfu/mL, at most 1×10⁴ cfu/mL, at most 1×10⁵cfu/mL, at most 1×10⁶ cfu/mL, at most 1×10⁷ cfu/mL, at most 1×10⁸cfu/mL, at most 1×10⁹ cfu/mL, or at most 1×10¹⁰ cfu/mL.

In still other aspects of this embodiment, a method disclosed herein mayqualitatively or quantitatively detect pathogen having a concentrationafter the enrichment step of f, e.g., about 1×10⁻⁵ cfu/mL to about1×10⁻⁴ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁻³ cfu/mL, about 1×10⁻⁵cfu/mL to about 1×10⁻² cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁻¹cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁰ cfu/mL, about 1×10⁻⁵ cfu/mLto about 1×10¹ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10² cfu/mL, about1×10⁻⁵ cfu/mL to about 1×10³ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁴cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁵ cfu/mL, about 1×10⁻⁵ cfu/mLto about 1×10⁶ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁷ cfu/mL, about1×10⁻⁵ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10⁹cfu/mL, about 1×10⁻⁵ cfu/mL to about 1×10¹⁰ cfu/mL, about 1×10⁻⁴ cfu/mLto about 1×10⁻³ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁻² cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10⁰ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻⁴cfu/mL to about 1×10² cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10³ cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10⁵ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻⁴cfu/mL to about 1×10⁷ cfu/mL, about 1×10⁻⁴ cfu/mL to about 1×10⁸ cfu/mL,about 1×10⁻⁴ cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻⁴ cfu/mL to about1×10¹⁰ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁻² cfu/mL, about 1×10⁻³cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁰cfu/mL, about 1×10⁻³ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻³ cfu/mLto about 1×10² cfu/mL, about 1×10⁻³ cfu/mL to about 1×10³ cfu/mL, about1×10⁻³ cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁵cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻³ cfu/mLto about 1×10⁷ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10⁸ cfu/mL, about1×10⁻³ cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻³ cfu/mL to about 1×10¹⁰cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁻¹ cfu/mL, about 1×10⁻² cfu/mLto about 1×10⁰ cfu/mL, about 1×10⁻² cfu/mL to about 1×10¹ cfu/mL, about1×10⁻² cfu/mL to about 1×10² cfu/mL, about 1×10⁻² cfu/mL to about 1×10³cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁴ cfu/mL, about 1×10⁻² cfu/mLto about 1×10⁵ cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁶ cfu/mL, about1×10⁻² cfu/mL to about 1×10⁷ cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁸cfu/mL, about 1×10⁻² cfu/mL to about 1×10⁹ cfu/mL, about 1×10⁻² cfu/mLto about 1×10¹⁰ cfu/mL, about 1×10¹ cfu/mL to about 1×10⁰ cfu/mL, about1×10¹ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10²cfu/mL, about 1×10¹ cfu/mL to about 1×10³ cfu/mL, about 1×10⁻¹ cfu/mL toabout 1×10⁴ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁻¹ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁻¹ cfu/mLto about 1×10⁹ cfu/mL, about 1×10⁻¹ cfu/mL to about 1×10¹⁰ cfu/mL, about1×10⁰ cfu/mL to about 1×10¹ cfu/mL, about 1×10⁰ cfu/mL to about 1×10²cfu/mL, about 1×10⁰ cfu/mL to about 1×10³ cfu/mL, about 1×10⁰ cfu/mL toabout 1×10⁴ cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁰ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁰ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁰ cfu/mL toabout 1×10⁹ cfu/mL, about 1×10⁰ cfu/mL to about 1×10¹⁰ cfu/mL, about1×10¹ cfu/mL to about 1×10² cfu/mL, about 1×10¹ cfu/mL to about 1×10³cfu/mL, about 1×10¹ cfu/mL to about 1×10⁴ cfu/mL, about 1×10¹ cfu/mL toabout 1×10⁵ cfu/mL, about 1×10¹ cfu/mL to about 1×10⁶ cfu/mL, about1×10¹ cfu/mL to about 1×10⁷ cfu/mL, about 1×10¹ cfu/mL to about 1×10⁸cfu/mL, about 1×10¹ cfu/mL to about 1×10⁹ cfu/mL, about 1×10¹ cfu/mL toabout 1×10¹⁰ cfu/mL, about 1×10² cfu/mL to about 1×10³ cfu/mL, about1×10² cfu/mL to about 1×10⁴ cfu/mL, about 1×10² cfu/mL to about 1×10⁵cfu/mL, about 1×10² cfu/mL to about 1×10⁶ cfu/mL, about 1×10² cfu/mL toabout 1×10⁷ cfu/mL, about 1×10² cfu/mL to about 1×10⁸ cfu/mL, about1×10² cfu/mL to about 1×10⁹ cfu/mL, about 1×10² cfu/mL to about 1×10¹⁰cfu/mL, about 1×10³ cfu/mL to about 1×10⁴ cfu/mL, about 1×10³ cfu/mL toabout 1×10⁵ cfu/mL, about 1×10³ cfu/mL to about 1×10⁶ cfu/mL, about1×10³ cfu/mL to about 1×10⁷ cfu/mL, about 1×10³ cfu/mL to about 1×10⁸cfu/mL, about 1×10³ cfu/mL to about 1×10⁹ cfu/mL, about 1×10³ cfu/mL toabout 1×10¹⁰ cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁵ cfu/mL, about1×10⁴ cfu/mL to about 1×10⁶ cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁷cfu/mL, about 1×10⁴ cfu/mL to about 1×10⁸ cfu/mL, about 1×10⁴ cfu/mL toabout 1×10⁹ cfu/mL, or about 1×10⁴ cfu/mL to about 1×10¹⁰ cfu/mL.

Common detection procedures used to determine the presence or absence ofa pathogen include nucleic acid-based detection methods, protein-baseddetection methods, activity-based detection methods, growth-baseddetection methods, and sensor-based detection methods.

In one embodiment, detection presence or absence of a pathogen ofinterest occurs using a nucleic acid-based detection method.Non-limiting examples of nucleic acid-based detection method includeDNA-based detection methods and RNA-based detection methods. DNA-baseddetection methods include, without limitation, like Southern blotanalysis, PCR-based assays, sequence analysis, immuno-based detectionassays, and hybridization assays using FRET, polarization or otherfluorescent, chemiluminescent or bioluminescent detection methods.RNA-based detection methods include, without limitation, like Northernblot analysis, RT-PCR-based assays, RNA sequencing, immuno-baseddetection assays, and hybridization assays using FRET, polarization orother fluorescent, chemiluminescent or bioluminescent detection methods.

In one embodiment, detection presence or absence of a pathogen ofinterest occurs using a protein-based detection method. Non-limitingexamples of protein-based detection methods include gel-based detectionmethods, immuno-based detection methods and protein-interaction-basedmethods. Gel-based detection methods include, without limitationpolyacrylamide gel electrophoresis and SDS-PAGE. Immuno-based detectionmethods include, without limitation, Western blot analysis, ELISA, andimmunoprecipitation. Protein-interaction-based methods include, withoutlimitation, protein-protein interaction-based assays, protein-DNAinteraction-based assays, and protein-RNA interaction-based assays.

In one embodiment, detection presence or absence of a pathogen ofinterest occurs using an activity-based detection method. Non-limitingexamples of activity-based detection methods include enzymatic activityassays and assays based on protein function. An enzymatic activityassays typically involves incubating an aliquot from an enrichment mediaor from the supernatant taken after an immunopurification step in abuffered solution containing a suitable substrate. If the desired enzymeis present in the aliquot, then is will catalyze the conversion of thesubstrate into a product. Measuring either the loss of substrate or theformation of product can then be either qualitatively or quantitativelycorrelated to the amount of enzyme present and hence the amount ofpathogen in the enrichment media. Similarly, assays based on proteinfunction measure the amount of function present in the sample andextrapolate the amount of pathogen in the enrichment media based on thismeasurement.

In one embodiment, detection presence or absence of a pathogen ofinterest occurs using a growth-based detection method. Non-limitingexamples of growth-based detection method include plating assaysmeasuring colony formation with or without growth selective agents andspectrophotometer assays measuring cell density. A plating assaytypically involves plating an aliquot of enrichment media onto an agarplate comprising nutrients to sustain growth of the pathogen ofinterest. The inoculated agar plates are then incubated for a specifiedtemperature and time and growth of pathogen colonies assessed. In someembodiments, the agar plates are incubated at about 25° C. to about 42°C. for about 12 hours to about 48 hours. In aspects of this embodiments,the agar plates are incubated at about 37° C. for about 14 hours toabout 16 hours. This assessment may be qualitative by simply assessingthe presence or absence of pathogen colonies, or quantitative, where thenumber of pathogen colonies are counted. Besides nutrients, an agarplate may contain a chromogenic compound that stains or otherwiseprovides a visual signal that identifies the colony as a pathogencolony. In addition, an agar plate may contain a compound that selectsfor pathogen colony growth by either supplying a compound thatfacilitates or is required for pathogen growth or inhibits the growth ofcontaminating microorganisms. A spectrophotometer assays measuring celldensity typically involves measuring the cell density of an aliquottaken from enrichment media using a spectrophotometer at a specificwavelength.

In one embodiment, detection presence or absence of a pathogen ofinterest occurs using a sensor-based detection method. Sensors can beclassified according to the type of energy being transfer, such as,e.g., thermal, electromagnetic, mechanical, and electrochemical.

In an aspect of this embodiment, a sensor-based detection method is abiosensor-based detection method. A biosensor is a type of analyticaldevice incorporating a biological material, a biologically derivedmaterial or a biomimic intimately associated with or integrated within aphysicochemical transducer or transducing microsystem. A biosensorconverts the modification of the physical or chemical properties of abiomatrix (e.g., enzyme, antibodies, receptors, organelles,microorganisms) into an electric or other kinds of signal whoseamplitude depends on the concentration of defined analytes in thesolution.

Non-limiting examples of a biosensor include an enzymatic biosensor, aDNA sensor, and an immunosensor. Enzyme-based biosensor require theimmobilization of an enzyme onto an electrode surface for thequantification of an analyte. DNA-based biosensor require theimmobilization of non-complimentary DNA strand of a target sequence ontoan electrode surface for the quantification of an analyte. Immuno-basedbiosensor require the immobilization of an antibody onto an electrodesurface for the quantification of an analyte.

A biosensor comprises a biorecognition element, a signal transducer, anda detector. The biorecognition element includes antibodies, peptides,nucleic acids, or enzymes and is the portion of the sensor thatinitially binds to or interacts with the analyte (e.g., a pathogen). Inmany cases this is associated with a conformational change, substratecleavage, or enzymatic reaction that transduces the biorecognition eventinto a signal that may be detected via several modalities. A biosensorcomprises two electrodes (reference and a working electrodes) or threeelectrodes (reference, working, and counter electrode).

A reference electrode includes liquid and solid-state referenceelectrodes and is an electrode having a known stable potential to whichall other electrode potentials are referenced. A reference electrode canbe manufactured by film deposition, electroplating and screen printing.Non-limiting examples of a reference electrode include silver-silverchloride electrode, calomel electrode, hydrogen electrode,mercury-mercury oxide electrode, mercury-mercurous sulfate electrode,copper-copper sulfate electrode, and palladium hydride electrode.

A biosensor typically uses the power of electrochemical techniques forbiological processes by quantitatively producing an electrical signalthat relates to the concentration of a biological analyte. Suchelectrochemical biosensors can be classified as potentiometric,amperometric, voltametric and impedimetric/conductimetric based upon theanalytical principles of operation. Potentiometric electrochemicalsensors measure an equilibrium potential difference between a workingelectrode and a reference electrode at zero current flow. Voltammetricelectrochemical sensors measure the current as a function of varyingpotential is applied between a working electrode and a referenceelectrode. Amperometric electrochemical sensors measure the current as afunction of constant potential is applied between a working electrodeand a reference electrode. Impedimetric/conductimetric electrochemicalsensors measure the changes of electrical properties between a workingelectrode and a reference electrode. Electrochemical detection using abiosensor may be a direct measurement of the change in electricalproperties or an indirect measurement using an auxiliary reaction whichinvolves a marking (redox active) compound for signal generation.

Aspect of the present specification disclose, in part, a detectionsolution. When a biosensor is a glucose-based biosensor, a detectionsolution disclosed herein typically includes a buffered solutioncomprises magnesium chloride, p-aminofenil phosphate, and glucose.Optionally, such a detection solution may comprise a surfactant asdisclosed herein.

Any buffer may be used, with the proviso that the resulting bufferedsolution is useful to practice the methods disclosed herein. A bufferedsolution can be varied as appropriate by one skilled in the art andgenerally depends, in part, on the pH value desired for the mobilephase, the protein being eluted, and the conductivity values beingemployed. Therefore, aspects of this embodiment may optionally include,e.g., 2-(N-morpholino) ethanesulfonic acid (MES),N-(2-acetamido)iminodiacetic acid (ADA), dimethylarsinic acid(Cacodylate), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), cholamine chloride,N,N′-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino) propanesulfonic acid (MOPS),2-{[tris(hydroxymethyl)methyl]amino} ethanesulfonic acid (TES),N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES),piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO),N-tris(hydroxymethyl) methylglycine (Tricine),tris(hydroxymethyl)methylamine (Tris), acetamidoglycine,N,N-bis(2-hydroxyethyl)glycine (Bicine),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS); acetate buffers,such as, e.g., magnesium acetate, potassium acetate, and Tris acetate;borate buffers; citrate buffers; phosphate buffers, such as, e.g.,potassium phosphate buffers and sodium phosphate buffers; salinebuffers, such as, e.g., phosphate-buffered saline (PBS), HEPES-bufferedsaline (HBS), and Tris-buffered saline (TBS), saline sodium citrate(SSC); universal buffers, such as, e.g., buffers comprising citric acidand potassium phosphate, Britton-Robinson buffer, Carmody buffer and thelike, or any combination thereof. Non-limiting examples of how to makeand use specific buffers are described in, e.g., MOLECULAR CLONING, ALABORATORY MANUAL (Joseph Sambrook & David W. Russell eds., Cold SpringHarbor Laboratory Press, 3rd ed. 2001) and CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons,2004).

Any concentration of magnesium chloride may be used in a detectionsolution, with the proviso that the concentration is useful to practicethe methods disclosed herein. In aspects of this embodiment, aconcentration of magnesium chloride used in a detection solution may be,e.g., about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM,about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM,about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In other aspectsof this embodiment, a concentration of magnesium chloride used in adetection solution may be, e.g., at least 5 mM, at least 10 mM, at least15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM,at least 40 mM, at least 45 mM, at least 50 mM, at least 55 mM, at least60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM,at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM. Inyet other aspects of this embodiment, a concentration of magnesiumchloride used in a detection solution may be, e.g., at most 5 mM, atmost 10 mM, at most 15 mM, at most 20 mM, at most 25 mM, at most 30 mM,at most 35 mM, at most 40 mM, at most 45 mM, at most 50 mM, at most 55mM, at most 60 mM, at most 65 mM, at most 70 mM, at most 75 mM, at most80 mM, at most 85 mM, at most 90 mM, at most 95 mM, or at most 100 mM.In still other aspects of this embodiment, a concentration of magnesiumchloride used in a detection solution may be, e.g., about 5 mM to about10 mM, about 5 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mMto about 40 mM, about 5 mM to about 50 mM, about 5 mM to about 60 mM,about 5 mM to about 70 mM, about 5 mM to about 80 mM, about 5 mM toabout 90 mM, about 5 mM to about 100 mM, about 10 mM to about 20 mM,about 10 mM to about 30 mM, about 10 mM to about 40 mM, about 10 mM toabout 50 mM, about 10 mM to about 60 mM, about 10 mM to about 70 mM,about 10 mM to about 80 mM, about 10 mM to about 90 mM, about 10 mM toabout 100 mM, about 20 mM to about 30 mM, about 20 mM to about 40 mM,about 20 mM to about 50 mM, about 20 mM to about 60 mM, about 20 mM toabout 70 mM, about 20 mM to about 80 mM, about 20 mM to about 90 mM, orabout 20 mM to about 100 mM.

Any concentration of p-aminofenil phosphate may be used in a detectionsolution, with the proviso that the concentration is useful to practicethe methods disclosed herein. In aspects of this embodiment, aconcentration of p-aminofenil phosphate used in a detection solution maybe, e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, or about2.5 mM. In other aspects of this embodiment, a concentration ofp-aminofenil phosphate used in a detection solution may be, e.g., atleast 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM, atleast 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, atleast 0.9 mM, at least 1.0 mM, at least 1.1 mM, at least 1.2 mM, atleast 1.3 mM, at least 1.4 mM, at least 1.5 mM, at least 1.6 mM, atleast 1.7 mM, at least 1.8 mM, at least 1.9 mM, at least 2.0 mM, atleast 2.1 mM, at least 2.2 mM, at least 2.3 mM, at least 2.4 mM, or atleast 2.5 mM. In yet other aspects of this embodiment, a concentrationof p-aminofenil phosphate used in a detection solution may be, e.g., atmost 0.1 mM, at most 0.2 mM, at most 0.3 mM, at most 0.4 mM, at most 0.5mM, at most 0.6 mM, at most 0.7 mM, at most 0.8 mM, at most 0.9 mM, atmost 1.0 mM, at most 1.1 mM, at most 1.2 mM, at most 1.3 mM, at most 1.4mM, at most 1.5 mM, at most 1.6 mM, at most 1.7 mM, at most 1.8 mM, atmost 1.9 mM, at most 2.0 mM, at most 2.1 mM, at most 2.2 mM, at most 2.3mM, at most 2.4 mM, or at most 2.5 mM. In still other aspects of thisembodiment, a concentration of p-aminofenil phosphate used in adetection solution may be, e.g., about 0.1 mM to about 0.5 mM, about 0.1mM to about 1.0 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about2.0 mM, about 0.1 mM to about 2.5 mM, about 0.3 mM to about 0.5 mM,about 0.3 mM to about 1.0 mM, about 0.3 mM to about 1.5 mM, about 0.3 mMto about 2.0 mM, about 0.3 mM to about 2.5 mM, about 0.5 mM to about 1.0mM, about 0.5 mM to about 1.5 mM, about 0.5 mM to about 2.0 mM, about0.5 mM to about 2.5 mM, about 0.7 mM to about 1.0 mM, about 0.7 mM toabout 1.5 mM, about 0.7 mM to about 2.0 mM, about 0.7 mM to about 2.5mM, about 1.0 mM to about 1.5 mM, about 1.0 mM to about 2.0 mM, about1.0 mM to about 2.5 mM, about 1.5 mM to about 2.0 mM, about 1.5 mM toabout 2.5 mM, or about 2.0 mM to about 2.5 mM.

Any concentration of glucose may be used in a detection solution, withthe proviso that the concentration is useful to practice the methodsdisclosed herein. In aspects of this embodiment, a concentration ofglucose used in a detection solution may be, e.g., about 1 mM, about 2mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM,about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. Inother aspects of this embodiment, a concentration of glucose used in adetection solution may be, e.g., at least 1 mM, at least 2 mM, at least3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, atleast 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 20mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, atleast 45 mM, or at least 50 mM. In yet other aspects of this embodiment,a concentration of glucose used in a detection solution may be, e.g., atmost 1 mM, at most 2 mM, at most 3 mM, at most 4 mM, at most 5 mM, atmost 6 mM, at most 7 mM, at most 8 mM, at most 9 mM, at most 10 mM, atmost 15 mM, at most 20 mM, at most 25 mM, at most 30 mM, at most 35 mM,at most 40 mM, at most 45 mM, or at most 50 mM. In still other aspectsof this embodiment, a concentration of glucose used in a detectionsolution may be, e.g., about 1 mM to about 10 mM, about 1 mM to about 15mM, about 1 mM to about 20 mM, about 1 mM to about 30 mM, about 1 mM toabout 40 mM, about 1 mM to about 50 mM, about 5 mM to about 10 mM, about5 mM to about 15 mM, about 5 mM to about 20 mM, about 5 mM to about 30mM, about 5 mM to about 40 mM, about 5 mM to about 50 mM, about 10 mM toabout 15 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM,about 10 mM to about 40 mM, or about 10 mM to about 50 mM.

Any concentration of surfactant may be used in a detection solution,with the proviso that the concentration is useful to practice themethods disclosed herein. Surfactants disclosed herein at theconcentrations disclosed herein may be used in preparing a detectionsolution. In aspects of this embodiment, a detection solution comprisesbetween 0.1% (v/v) and 10% (v/v) detergent B-Per, or a surfactant ofpolyoxyethylene glycol sorbitan alkyl esters family (TWEEN® family) orpolyoxyethylene glycol octylphenol ethers family (TRITON® family).

In an aspect of this embodiment, a detection solution may be 200 mMPhosphate buffer (pH 5.5) detection solution. In an aspect of thisembodiment, 200 mM Phosphate buffer (pH 5.5) detection solution maycomprise 0.0021 g/mL Sodium Hydrogen Phosphate, 0.0289 g/mL SodiumDihydrogen Phosphate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mLGlucose, and 0.0002 g/mL p-aminophenyl phosphate.

In an aspect of this embodiment, a detection solution may be 200 mMPhosphate buffer (pH 5.7) detection solution. In an aspect of thisembodiment, 200 mM Phosphate buffer (pH 5.7) detection solution maycomprise 0.0174 g/mL Potassium Hydrogen Phosphate, 0.0204 g/mL PotassiumPhosphate (Phtalate), 0.0020 g/mL Magnesium chloride, 0.0018 g/mLGlucose, and 0.0002 g/mL p-aminophenyl phosphate.

In an aspect of this embodiment, a detection solution may be 200 mMPhosphate buffer (pH 7.0) detection solution. In an aspect of thisembodiment, 200 mM Phosphate buffer (pH 7.0) detection solution maycomprise 0.0156 g/mL Sodium Hydrogen Phosphate, 0.0142 g/mL SodiumDihydrogen Phosphate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mLGlucose, and 0.0002 g/mL p-aminophenyl phosphate.

In an aspect of this embodiment, a detection solution may be 160 mMPhosphate-citrate buffer (pH 5.5) detection solution. In an aspect ofthis embodiment, 160 mM Phosphate-citrate buffer (pH 5.5) detectionsolution may comprise 0.0161 g/mL Sodium Hydrogen Phosphate, 0.0090 g/mLCitric Acid, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and0.0002 g/mL p-aminophenyl phosphate.

In an aspect of this embodiment, a detection solution may be 200 mMAcetate buffer (pH 5.7) detection solution. In an aspect of thisembodiment, 200 mM Acetate buffer (pH 5.7) detection solution maycomprise 1.15 μL Acetic Acid, 0.0015 g/mL Sodium Acetate, 0.0020 g/mLMagnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenylphosphate.

In an aspect of this embodiment, a detection solution may be 200 mM Trisbuffer (pH 9.8) detection solution. In an aspect of this embodiment, 200mM Tris buffer (pH 9.8) detection solution may comprise 0.0243 g/mLTRIS, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002g/mL p-aminophenyl phosphate.

Aspect of the present specification disclose, in part, incubation of adetection solution. Incubation of a detection solution is performedunder temperature and time parameters that facilitate detection of asignal. Incubation a detection solution may be performed underagitation/rotation.

Any temperature may be used during incubation of a detection solution,with the proviso that the temperature is useful to practice the methodsdisclosed herein. In aspects of this embodiment, a temperature used toincubate a detection solution may be, e.g., about 15° C., about 16° C.,about 17° C., about 18° C., about 19° C., about 20° C., about 21° C.,about 22° C., about 23° C., about 24° C., or about 25° C. In otheraspects of this embodiment, a temperature used to incubate a detectionsolution may be, e.g., at least 15° C., at least 16° C., at least 17°C., at least 18° C., at least 19° C., at least 20° C., at least 21° C.,at least 22° C., at least 23° C., at least 24° C., or at least 25° C. Inyet other aspects of this embodiment, a temperature used to incubate adetection solution may be, e.g., at most 15° C., at most 16° C., at most17° C., at most 18° C., at most 19° C., at most 20° C., at most 21° C.,at most 22° C., at most 23° C., at most 24° C., or at most 25° C. Instill other aspects of this embodiment, a temperature used to incubateimmunmagnetic particles with enrichment media comprising the pathogen ofinterest may be, e.g., about 15° C. to about 19° C., about 16° C. toabout 20° C., about 17° C. to about 21° C., about 18° C. to about 22°C., about 19° C. to about 23° C., about 20° C. to about 24° C., about21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. toabout 27° C., about 24° C. to about 28° C., or about 25° C. to about 29°C.

Any time may be used during incubation of a detection solution, with theproviso that the time is useful to practice the methods disclosedherein. In aspects of this embodiment, a time used to incubate adetection solution may be, e.g., about 5 minutes, about 10 minutes,about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes,about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes,about 90 minutes, about 100 minutes, about 110 minutes, about 120minutes, about 130 minutes, about 140 minutes, or about 150 minutes. Inother aspects of this embodiment, a time used to incubate a detectionsolution may be, e.g., at least 5 minutes, at least 10 minutes, at least15 minutes, at least 20 minutes, at least 30 minutes, at least 40minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes,at least 80 minutes, at least 90 minutes, at least 100 minutes, at least110 minutes, at least 120 minutes, at least 130 minutes, at least 140minutes, or at least 150 minutes. In yet other aspects of thisembodiment, a time used to a detection solution may be, e.g., at most 5minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, atmost 30 minutes, at most 40 minutes, at most 50 minutes, at most 60minutes, at most 70 minutes, at most 80 minutes, at most 90 minutes, atmost 100 minutes, at most 110 minutes, at most 120 minutes, at most 130minutes, at most 140 minutes, or at most 150 minutes.

In yet other aspects of this embodiment, a time used to a detectionsolution may be, e.g., about 5 minutes to about 20 minutes, about 5minutes to about 30 minutes, about 5 minutes to about 40 minutes, about5 minutes to about 50 minutes, about 5 minutes to about 60 minutes,about 5 minutes to about 70 minutes, about 5 minutes to about 80minutes, about 5 minutes to about 90 minutes, about 5 minutes to about100 minutes, about 5 minutes to about 110 minutes, about 5 minutes toabout 120 minutes, about 5 minutes to about 130 minutes, about 5 minutesto about 140 minutes, about 5 minutes to about 150 minutes, about 10minutes to about 20 minutes, about 10 minutes to about 30 minutes, about10 minutes to about 40 minutes, about 10 minutes to about 50 minutes,about 10 minutes to about 60 minutes, about 10 minutes to about 70minutes, about 10 minutes to about 80 minutes, about 10 minutes to about90 minutes, about 10 minutes to about 100 minutes, about 10 minutes toabout 110 minutes, about 10 minutes to about 120 minutes, about 10minutes to about 130 minutes, about 10 minutes to about 140 minutes,about 10 minutes to about 150 minutes, about 20 minutes to about 30minutes, about 20 minutes to about 40 minutes, about 20 minutes to about50 minutes, about 20 minutes to about 60 minutes, about 20 minutes toabout 70 minutes, about 20 minutes to about 80 minutes, about 20 minutesto about 90 minutes, about 20 minutes to about 100 minutes, about 20minutes to about 110 minutes, about 20 minutes to about 120 minutes,about 20 minutes to about 130 minutes, about 20 minutes to about 140minutes, about 20 minutes to about 150 minutes, about 30 minutes toabout 40 minutes, about 30 minutes to about 50 minutes, about 30 minutesto about 60 minutes, about 30 minutes to about 70 minutes, about 30minutes to about 80 minutes, about 30 minutes to about 90 minutes, about30 minutes to about 100 minutes, about 30 minutes to about 110 minutes,about 30 minutes to about 120 minutes, about 30 minutes to about 130minutes, about 30 minutes to about 140 minutes, about 30 minutes toabout 150 minutes, about 60 minutes to about 70 minutes, about 60minutes to about 80 minutes, about 60 minutes to about 90 minutes, about60 minutes to about 100 minutes, about 60 minutes to about 110 minutes,about 60 minutes to about 120 minutes, about 60 minutes to about 130minutes, about 60 minutes to about 140 minutes, about 60 minutes toabout 150 minutes, about 90 minutes to about 100 minutes, about 90minutes to about 110 minutes, about 90 minutes to about 120 minutes,about 90 minutes to about 130 minutes, about 90 minutes to about 140minutes, about 90 minutes to about 150 minutes, about 120 minutes toabout 130 minutes, about 120 minutes to about 140 minutes, or about 120minutes to about 150 minutes.

In aspects of this embodiment, a detection solution may be incubated ata temperature of, e.g., about 15° C., about 16° C., about 17° C., about18° C., about 19° C., about 20° C., about 21° C., about 22° C., about23° C., about 24° C., or about 25° C. for a time of, e.g., about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70minutes, about 80 minutes, about 90 minutes, about 100 minutes, about110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, orabout 150 minutes.

In other aspects of this embodiment, a detection solution may beincubated at a temperature of, e.g., at least 15° C., at least 16° C.,at least 17° C., at least 18° C., at least 19° C., at least 20° C., atleast 21° C., at least 22° C., at least 23° C., at least 24° C., or atleast 25° C. for a time of, e.g., at least 5 minutes, at least 10minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes,at least 40 minutes, at least 50 minutes, at least 60 minutes, at least70 minutes, at least 80 minutes, at least 90 minutes, at least 100minutes, at least 110 minutes, at least 120 minutes, at least 130minutes, at least 140 minutes, or at least 150 minutes.

In yet other aspects of this embodiment, a detection solution may beincubated at a temperature of, e.g., at most 15° C., at most 16° C., atmost 17° C., at most 18° C., at most 19° C., at most 20° C., at most 21°C., at most 22° C., at most 23° C., at most 24° C., or at most 25° C.,for a time of, e.g., at most 5 minutes, at most 10 minutes, at most 15minutes, at most 20 minutes, at most 30 minutes, at most 40 minutes, atmost 50 minutes, at most 60 minutes, at most 70 minutes, at most 80minutes, at most 90 minutes, at most 100 minutes, at most 110 minutes,at most 120 minutes, at most 130 minutes, at most 140 minutes, or atmost 150 minutes.

In still other aspects of this embodiment, a detection solution may beincubated at a temperature of, e.g., about 15° C. to about 19° C., about16° C. to about 20° C., about 17° C. to about 21° C., about 18° C. toabout 22° C., about 19° C. to about 23° C., about 20° C. to about 24°C., about 21° C. to about 25° C., about 22° C. to about 26° C., about23° C. to about 27° C., about 24° C. to about 28° C., or about 25° C. toabout 29° C. for a time of, e.g., about 5 minutes to about 20 minutes,about 5 minutes to about 30 minutes, about 5 minutes to about 40minutes, about 5 minutes to about 50 minutes, about 5 minutes to about60 minutes, about 5 minutes to about 70 minutes, about 5 minutes toabout 80 minutes, about 5 minutes to about 90 minutes, about 5 minutesto about 100 minutes, about 5 minutes to about 110 minutes, about 5minutes to about 120 minutes, about 5 minutes to about 130 minutes,about 5 minutes to about 140 minutes, about 5 minutes to about 150minutes, about 10 minutes to about 20 minutes, about 10 minutes to about30 minutes, about 10 minutes to about 40 minutes, about 10 minutes toabout 50 minutes, about 10 minutes to about 60 minutes, about 10 minutesto about 70 minutes, about 10 minutes to about 80 minutes, about 10minutes to about 90 minutes, about 10 minutes to about 100 minutes,about 10 minutes to about 110 minutes, about 10 minutes to about 120minutes, about 10 minutes to about 130 minutes, about 10 minutes toabout 140 minutes, about 10 minutes to about 150 minutes, about 20minutes to about 30 minutes, about 20 minutes to about 40 minutes, about20 minutes to about 50 minutes, about 20 minutes to about 60 minutes,about 20 minutes to about 70 minutes, about 20 minutes to about 80minutes, about 20 minutes to about 90 minutes, about 20 minutes to about100 minutes, about 20 minutes to about 110 minutes, about 20 minutes toabout 120 minutes, about 20 minutes to about 130 minutes, about 20minutes to about 140 minutes, about 20 minutes to about 150 minutes,about 30 minutes to about 40 minutes, about 30 minutes to about 50minutes, about 30 minutes to about 60 minutes, about 30 minutes to about70 minutes, about 30 minutes to about 80 minutes, about 30 minutes toabout 90 minutes, about 30 minutes to about 100 minutes, about 30minutes to about 110 minutes, about 30 minutes to about 120 minutes,about 30 minutes to about 130 minutes, about 30 minutes to about 140minutes, about 30 minutes to about 150 minutes, about 60 minutes toabout 70 minutes, about 60 minutes to about 80 minutes, about 60 minutesto about 90 minutes, about 60 minutes to about 100 minutes, about 60minutes to about 110 minutes, about 60 minutes to about 120 minutes,about 60 minutes to about 130 minutes, about 60 minutes to about 140minutes, about 60 minutes to about 150 minutes, about 90 minutes toabout 100 minutes, about 90 minutes to about 110 minutes, about 90minutes to about 120 minutes, about 90 minutes to about 130 minutes,about 90 minutes to about 140 minutes, about 90 minutes to about 150minutes, about 120 minutes to about 130 minutes, about 120 minutes toabout 140 minutes, or about 120 minutes to about 150 minutes.

After incubation of a detection solution, an aliquot is removed foranalysis of a detectable electrochemical signal. In aspects of thisembodiment, a volume of an aliquot to be analyzed for a detectableelectrochemical signal may be, e.g., about 1 μL, about 2 μL, about 3 μL,about 4 μL, about 5 μL, about 6 μL, about 7 μL, about 8 μL, about 9 μL,about 10 μL, about 11 μL, about 12 μL, about 13 μL, about 14 μL, about15 μL, about 16 μL, about 17 μL, about 18 μL, about 19 μL, or about 20μL. In other aspects of this embodiment, a volume of an aliquot to beanalyzed for a detectable electrochemical signal may be, e.g., at least1 μL, at least 2 μL, at least 3 μL, at least 4 μL, at least 5 μL, atleast 6 μL, at least 7 μL, at least 8 μL, at least 9 μL, at least 10 μL,at least 11 μL, at least 12 μL, at least 13 μL, at least 14 μL, at least15 μL, at least 16 μL, at least 17 μL, at least 18 μL, at least 19 μL,or at least 20 μL. In yet other aspects of this embodiment, a volume ofan aliquot to be analyzed for a detectable electrochemical signal maybe, e.g., at most 1 μL, at most 2 μL, at most 3 μL, at most 4 μL, atmost 5 μL, at most 6 μL, at most 7 μL, at most 8 μL, at most 9 μL, atmost 10 μL, at most 11 μL, at most 12 μL, at most 13 μL, at most 14 μL,at most 15 μL, at most 16 μL, at most 17 μL, at most 18 μL, at most 19μL, or at most 20 μL. In yet other aspects of this embodiment, a volumeof an aliquot to be analyzed for a detectable electrochemical signal maybe, e.g., about 1 μL to about 5 μL, about 1 μL to about 10 μL, about 1μL to about 15 μL, about 1 μL to about 20 μL, 2 μL to about 5 μL, about2 μL to about 10 μL, about 2 μL to about 15 μL, about 2 μL to about 20μL, about 5 μL to about 10 μL, about 5 μL to about 15 μL, about 5 μL toabout 20 μL, about 10 μL to about 15 μL, about 10 μL to about 20 μL, orabout 15 μL to about 20 μL.

An electrochemical signal can be analyzed using an instrument which iscapable of measuring and/or analyzing potentiometric, voltammetric,amperometric and/or impedance/conductance parameters. Typically, suchinstrumentation is operated using computer-controlled software. Anon-limiting example is PalmSens3, a potentiostat, galvanostat, andimpedance analyzer and PSTrace, its accompanying software (PalmSens BV,Utrecht, Netherlands).

In other embodiments, a method disclosed herein can be performed tocompletion in, e.g., about 18 hours, about 20 hours, about 22 hours,about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32hours, about 34 hours, or about 36 hours. In yet other embodiments, amethod comprising a pre-enrichment step disclosed herein and anenrichment step disclosed herein can be performed to completion in,e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours,about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34hours, or about 36 hours. In still other embodiments, a methodcomprising a pre-enrichment step disclosed herein, an enrichment stepdisclosed herein and a detection step disclosed herein can be performedto completion in, e.g., about 18 hours, about 20 hours, about 22 hours,about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32hours, about 34 hours, or about 36 hours. In other embodiments, a methodcomprising a pre-enrichment step disclosed herein, an enrichment stepdisclosed herein, a purification step disclosed herein, and a detectionstep disclosed herein can be performed to completion in, e.g., about 18hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours,about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about36 hours.

In other embodiments, a method disclosed herein can be performed tocompletion in, e.g., less than 18 hours, less than 20 hours, less than22 hours, less than 24 hours, less than 26 hours, less than 28 hours,less than 30 hours, less than 32 hours, less than 34 hours, or less than36 hours. In yet other embodiments, a method comprising a pre-enrichmentstep disclosed herein and an enrichment step disclosed herein can beperformed to completion in, e.g., less than 18 hours, less than 20hours, less than 22 hours, less than 24 hours, less than 26 hours, lessthan 28 hours, less than 30 hours, less than 32 hours, less than 34hours, or less than 36 hours. In still other embodiments, a methodcomprising a pre-enrichment step disclosed herein, an enrichment stepdisclosed herein and a detection step disclosed herein can be performedto completion in, e.g., less than 18 hours, less than 20 hours, lessthan 22 hours, less than 24 hours, less than 26 hours, less than 28hours, less than 30 hours, less than 32 hours, less than 34 hours, orless than 36 hours. In other embodiments, a method comprising apre-enrichment step disclosed herein, an enrichment step disclosedherein, a purification step disclosed herein, and a detection stepdisclosed herein can be performed to completion in, e.g., less than 18hours, less than 20 hours, less than 22 hours, less than 24 hours, lessthan 26 hours, less than 28 hours, less than 30 hours, less than 32hours, less than 34 hours, or less than 36 hours.

In other embodiments, a method disclosed herein can be performed tocompletion in, e.g., about 18 hours to about 20 hours, about 18 hours toabout 22 hours, about 18 hours to about 24 hours, about 18 hours toabout 26 hours, about 18 hours to about 28 hours, about 18 hours toabout 30 hours, about 18 hours to about 32 hours, about 18 hours toabout 34 hours, about 18 hours to about 36 hours, about 20 hours toabout 22 hours, about 20 hours to about 24 hours, about 20 hours toabout 26 hours, about 20 hours to about 28 hours, about 20 hours toabout 30 hours, about 20 hours to about 32 hours, about 20 hours toabout 34 hours, about 20 hours to about 36 hours, about 22 hours toabout 24 hours, about 22 hours to about 26 hours, about 22 hours toabout 28 hours, about 22 hours to about 30 hours, about 22 hours toabout 32 hours, about 22 hours to about 34 hours, about 22 hours toabout 36 hours, about 24 hours to about 26 hours, about 24 hours toabout 28 hours, about 24 hours to about 30 hours, about 24 hours toabout 32 hours, about 24 hours to about 34 hours, about 24 hours toabout 36 hours, about 26 hours to about 28 hours, about 26 hours toabout 30 hours, about 26 hours to about 32 hours, about 26 hours toabout 34 hours, about 26 hours to about 36 hours, about 28 hours toabout 30 hours, about 28 hours to about 32 hours, about 28 hours toabout 34 hours, about 28 hours to about 36 hours, about 30 hours toabout 32 hours, about 30 hours to about 34 hours, about 30 hours toabout 36 hours, about 32 hours to about 34 hours, about 32 hours toabout 36 hours, or about 34 hours to about 36 hours.

In yet other embodiments, a method comprising a pre-enrichment stepdisclosed herein and an enrichment step disclosed herein can beperformed to completion in, e.g., about 18 hours to about 20 hours,about 18 hours to about 22 hours, about 18 hours to about 24 hours,about 18 hours to about 26 hours, about 18 hours to about 28 hours,about 18 hours to about 30 hours, about 18 hours to about 32 hours,about 18 hours to about 34 hours, about 18 hours to about 36 hours,about 20 hours to about 22 hours, about 20 hours to about 24 hours,about 20 hours to about 26 hours, about 20 hours to about 28 hours,about 20 hours to about 30 hours, about 20 hours to about 32 hours,about 20 hours to about 34 hours, about 20 hours to about 36 hours,about 22 hours to about 24 hours, about 22 hours to about 26 hours,about 22 hours to about 28 hours, about 22 hours to about 30 hours,about 22 hours to about 32 hours, about 22 hours to about 34 hours,about 22 hours to about 36 hours, about 24 hours to about 26 hours,about 24 hours to about 28 hours, about 24 hours to about 30 hours,about 24 hours to about 32 hours, about 24 hours to about 34 hours,about 24 hours to about 36 hours, about 26 hours to about 28 hours,about 26 hours to about 30 hours, about 26 hours to about 32 hours,about 26 hours to about 34 hours, about 26 hours to about 36 hours,about 28 hours to about 30 hours, about 28 hours to about 32 hours,about 28 hours to about 34 hours, about 28 hours to about 36 hours,about 30 hours to about 32 hours, about 30 hours to about 34 hours,about 30 hours to about 36 hours, about 32 hours to about 34 hours,about 32 hours to about 36 hours, or about 34 hours to about 36 hours.

In still other embodiments, a method comprising a pre-enrichment stepdisclosed herein, an enrichment step disclosed herein and a detectionstep disclosed herein can be performed to completion in, e.g., about 18hours to about 20 hours, about 18 hours to about 22 hours, about 18hours to about 24 hours, about 18 hours to about 26 hours, about 18hours to about 28 hours, about 18 hours to about 30 hours, about 18hours to about 32 hours, about 18 hours to about 34 hours, about 18hours to about 36 hours, about 20 hours to about 22 hours, about 20hours to about 24 hours, about 20 hours to about 26 hours, about 20hours to about 28 hours, about 20 hours to about 30 hours, about 20hours to about 32 hours, about 20 hours to about 34 hours, about 20hours to about 36 hours, about 22 hours to about 24 hours, about 22hours to about 26 hours, about 22 hours to about 28 hours, about 22hours to about 30 hours, about 22 hours to about 32 hours, about 22hours to about 34 hours, about 22 hours to about 36 hours, about 24hours to about 26 hours, about 24 hours to about 28 hours, about 24hours to about 30 hours, about 24 hours to about 32 hours, about 24hours to about 34 hours, about 24 hours to about 36 hours, about 26hours to about 28 hours, about 26 hours to about 30 hours, about 26hours to about 32 hours, about 26 hours to about 34 hours, about 26hours to about 36 hours, about 28 hours to about 30 hours, about 28hours to about 32 hours, about 28 hours to about 34 hours, about 28hours to about 36 hours, about 30 hours to about 32 hours, about 30hours to about 34 hours, about 30 hours to about 36 hours, about 32hours to about 34 hours, about 32 hours to about 36 hours, or about 34hours to about 36 hours.

In other embodiments, a method comprising a pre-enrichment stepdisclosed herein, an enrichment step disclosed herein, a purificationstep disclosed herein, and a detection step disclosed herein can beperformed to completion in, e.g., about 18 hours to about 20 hours,about 18 hours to about 22 hours, about 18 hours to about 24 hours,about 18 hours to about 26 hours, about 18 hours to about 28 hours,about 18 hours to about 30 hours, about 18 hours to about 32 hours,about 18 hours to about 34 hours, about 18 hours to about 36 hours,about 20 hours to about 22 hours, about 20 hours to about 24 hours,about 20 hours to about 26 hours, about 20 hours to about 28 hours,about 20 hours to about 30 hours, about 20 hours to about 32 hours,about 20 hours to about 34 hours, about 20 hours to about 36 hours,about 22 hours to about 24 hours, about 22 hours to about 26 hours,about 22 hours to about 28 hours, about 22 hours to about 30 hours,about 22 hours to about 32 hours, about 22 hours to about 34 hours,about 22 hours to about 36 hours, about 24 hours to about 26 hours,about 24 hours to about 28 hours, about 24 hours to about 30 hours,about 24 hours to about 32 hours, about 24 hours to about 34 hours,about 24 hours to about 36 hours, about 26 hours to about 28 hours,about 26 hours to about 30 hours, about 26 hours to about 32 hours,about 26 hours to about 34 hours, about 26 hours to about 36 hours,about 28 hours to about 30 hours, about 28 hours to about 32 hours,about 28 hours to about 34 hours, about 28 hours to about 36 hours,about 30 hours to about 32 hours, about 30 hours to about 34 hours,about 30 hours to about 36 hours, about 32 hours to about 34 hours,about 32 hours to about 36 hours, or about 34 hours to about 36 hours.

Aspects of the present specification disclose, in part, a pathogenanalysis kit to carry out the method of detecting a pathogen disclosedherein. A pathogen analysis kit disclosed herein contains componentsnecessary for the detection of the pathogen of interest. In someaspects, a pathogen analysis kit disclosed herein components necessaryfor the detection of a single pathogen of interest. In some aspects, apathogen analysis kit disclosed herein components necessary for thedetection of a plurality of pathogens of interest.

In some embodiment, a pathogen analysis kit disclosed herein typicallycomprises a pre-enrichment media and an enrichment media. In someembodiment, a pathogen analysis kit disclosed herein typically comprisesa pre-enrichment media, an enrichment media, and a detection solution.In some embodiment, a pathogen analysis kit disclosed herein typicallycomprises a pre-enrichment media, an enrichment media, a detectionsolution and an electrochemical biosensor.

In some embodiment, a pathogen analysis kit disclosed herein typicallycomprises a pre-enrichment media, an enrichment media, and immunmagneticparticles capable of binding to a pathogen of interest. In someembodiment, a pathogen analysis kit disclosed herein typically comprisesa pre-enrichment media, an enrichment media, immunmagnetic particlescapable of binding to a pathogen of interest, and a magnetic source usedto capture the immunoparticles. In some embodiment, a pathogen analysiskit disclosed herein typically comprises a pre-enrichment media, anenrichment media, immunmagnetic particles capable of binding to apathogen of interest, and a detection solution. In some embodiment, apathogen analysis kit disclosed herein typically comprises apre-enrichment media, an enrichment media, immunmagnetic particlescapable of binding to a pathogen of interest, a magnetic source used tocapture the immunoparticles, and a detection solution. In someembodiment, a pathogen analysis kit disclosed herein typically comprisesa pre-enrichment media, an enrichment media, immunmagnetic particlescapable of binding to a pathogen of interest, a detection solution, andan electrochemical biosensor. In some embodiment, a pathogen analysiskit disclosed herein typically comprises a pre-enrichment media, anenrichment media, immunmagnetic particles capable of binding to apathogen of interest, a detection solution, a magnetic source used tocapture the immunoparticles, and an electrochemical biosensor.

A pathogen analysis kit disclosed herein may further comprise aninstrument which is capable of measuring and/or analyzingpotentiometric, voltammetric, amperometric and/or impedance/conductanceparameters.

A pathogen analysis kit disclosed herein may further comprise a suitablecontainer, for example, a vessel, vials, tubes, mini- or microfugetubes, test tube, flask, bottle, syringe or other container. Where anadditional component or agent is provided, the kit can contain one ormore additional containers into which this agent or component may beplaced. Kits herein will also typically include a means for containingthe agent (e.g. a vessel), composition and any other reagent containersin close confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

A pathogen analysis kit disclosed herein may further comprise a labelsor inserts. Labels or inserts include “printed matter,” e.g., paper orcardboard, or separate or affixed to a component, a kit or packingmaterial (e.g., a box), or attached to an ampule, tube or vialcontaining a kit component. Labels or inserts can additionally include acomputer readable medium, such as a disk (e.g., hard disk, flashmemory), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetictape, or an electrical storage media such as RAM and ROM or hybrids ofthese such as magnetic/optical storage media, FLASH media or memory typecards. Labels or inserts may include identifying information of one ormore components therein, amounts used for one or more componentstherein, step by step instructions of how to perform a method ofdetecting a pathogen of interest. Labels or inserts can includeinformation identifying manufacturer information, lot numbers,manufacturer location and date and patent information.

Aspects of the present specification may also be described as follows:

-   1. A method of detecting a pathogen in a sample the method    comprising the steps: a) incubation of the sample in a    pre-enrichment comprising a low growth nutrient component, a growth    inhibiting agent, and a surfactant with bacteriostatic or    bactericidal action, wherein the incubation at 37° C. for about 6    hours to about 24 hours; b) incubating an aliquot of pre-enrichment    media from step (a) in an enrichment media, the enrichment media    comprising a high growth nutrient component and ferric ammonium    citrate, wherein the incubation at about 15° C. to about 50° C. for    about 2 hours to about 24 hours; and c) detecting the presence of    absence of a pathogen by analyzing an aliquot of enrichment media    from step (b).-   2. The method according to embodiment 1, wherein the low growth    nutrient component is a peptone.-   3. The method according to embodiment 2, wherein the peptone is a    peptone from an animal source or a peptone from a plant source.-   4. The method according to embodiment 3, wherein the peptone from an    animal source is an acid casein peptone, a bacteriological peptone,    a beef extract powder, a casein peptone, a casein cc peptone, a    gelatin peptone, a meat peptone, a polypeptone proteose peptone, or    a proteose peptone No 3.-   5. The method according to embodiment 3, wherein the peptone from a    plant source is a malt extract, a soya peptone, or a yeast extract.-   6. The method according to any one of embodiments 1-5, wherein the    surfactant is at a concentration of about 0.001 (v/v) to about 10.0%    (v/v).-   7. The method according to any one of embodiments 1-6, wherein the    surfactant is an ionic surfactant, a zwitterionic (amphoteric)    surfactant, or a non-ionic surfactant.-   8. The method according to embodiments 7, wherein the ionic    surfactant is an anion surfactant or cationic surfactant.-   9. The method according to embodiments 8, wherein the anionic    surfactant is an alkyl sulfate, an alkyl ether sulfate, a docusate,    a sulfonate fluorosurfactant, an alkyl benzene sulfonate, an alkyl    aryl ether phosphate, an alkyl ether phosphate, an; alkyl    carboxylate, a sodium lauroyl sarcosinate, or a carboxylate    fluorosurfactant.-   10. The method according to embodiments 8, wherein the cationic    surfactant is an alkyltrimethylammonium salt, cetylpyridinium    chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium    chloride (BAC), benzethonium chloride (BZT),    5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride,    dioctadecyldimethylammonium bromide (DODAB), a pH-dependent primary    amine, a pH-dependent secondary amine, or a pH-dependent tertiary    amine.-   11. The method according to embodiments 7, wherein the zwitterionic    surfactant is 3-[(3-cholamidopropyl)    dimethylammonio]-1-propanesulfonate (CHAPS), a sultaine, a, betaine,    or a lecithin.-   12. The method according to embodiments 7, wherein the non-ionic    surfactant is a polyoxyethylene glycol sorbitan alkyl ester, a    poloxamer, an alkyl phenol polyglycol ether, a polyethylene glycol    alkyl aryl ether, a polyoxyethylene glycol alkyl ether,    2-dodecoxyethanol, a polyoxyethylene glycol octyl phenol ether, a    nonylphenol ethoxylate, a polyoxyethylene glycol alkylphenol ether,    a phenoxypolyethoxylethanol, a glucoside alkyl ether, a maltoside    alkyl ether, a thioglucoside alkyl ether, a digitonin, a glycerol    alkyl ester, an alkyl aryl polyether sulfate, an alcohol sulfonate,    a sorbitan alkyl ester, a cocamide ethanolamine, sucrose    monolaurate, dodecyl dimethylamine oxide, or sodium cholate.-   13. The method according to any one of embodiments 1-6, where the    surfactant is tetradecyl sodium dodecyl sulfate, octadecyl sulphate,    or 7-ethyl-2-methyl-4-undecyl sodium sulphate.-   14. The method according to any one of embodiments 1-13, where the    growth inhibiting agent is at a concentration of about 0.001 (v/v)    to about 10.0% (v/v).-   15. The method according to any one of embodiments 1-14, wherein the    growth inhibiting agent is a triarylmethane dye.-   16. The method according to embodiment 15, where the triarylmethane    dye is a methyl violet dye, a fuchsine dye, a phenol dye, or a    malachite green dye.-   17. The method according to embodiment 16, where the methyl violet    dye is methyl violet 2B, methyl violet 6B, or methyl violet 10B.-   18. The method according to embodiment 16, where the fuchsine dye is    pararosaniline, fuchsine, new fuchsine, fuchsin basic violet, or    fuchine acid.-   19. The method according to embodiment 16, where the phenol dye is    phenol red, chlorophenol red, or cresol red.-   20. The method according to embodiment 16, where the malachite green    dye is malachite green or brilliant green.-   21. The method according to embodiment 15, where the triarylmethane    dye is aluminon, aniline Blue WS, aurin, aurintricarboxylic acid,    brilliant blue FCF, brilliant green, bromocresol green, bromocresol    purple, bromophenol blue, bromopyrogallol red, bromothymol blue,    bromsulphthalein, chlorophenol red, coomassie brilliant blue, cresol    red, crystal violet, crystal violet lactone, ethyl green, fast green    FCF, fluoran, fuchsine, fuchsine acid, fuchsin basic violet,    gentian, green S, light green SF yellowish, malachite green, methyl    blue, methyl violet, new fuchsine, pararosaniline, patent blue V,    phenol red, phenolphthalein, rose bengal, thymolphthalein, victoria    blue BO, water blue, xylene cyanol, or xylenol orange.-   22. The method according to any one of embodiments 1-22, wherein the    pre-enrichment media further comprises a growth enhancing agent.-   23. The method according to embodiment 22, wherein the growth    enhancing agent is a siderophore.-   24. The method according to embodiment 23, wherein the siderophore    is Aerobactin, Alcaligin, Azotobactin, Bacillibactin,    Desferrioxamine B, Desferrioxamine E, Enterobactin, Ferrichrome,    Ferrioxiamina-B, Ferrioxiamina-E, Fusarinine C, Mycobactin,    Ornibactin, Petrobactin, Pyoverdine, Pyochelin, Salmochelin,    Staphyloferring A, Vibriobactin, or Yersiniabactin.-   25. The method according to any one of embodiments 1-24, wherein the    aliquot of pre-enrichment media in step (b) is about 1/50 to about    1/500 a volume of an enrichment media used in step (b).-   26. The method according to any one of embodiments 1-25, wherein the    high growth nutrient component is a peptone.-   27. The method according to embodiment 26, wherein the peptone is a    peptone from an animal source or a peptone from a plant source.-   28. The method according to embodiment 27, wherein the peptone from    an animal source is an acid casein peptone, a bacteriological    peptone, a beef extract powder, a casein peptone, a casein cc    peptone, a gelatin peptone, a meat peptone, a polypeptone proteose    peptone, or a proteose peptone No 3.-   29. The method according to embodiment 27, wherein the peptone from    a plant source is a malt extract, a soya peptone, or a yeast    extract.-   30. The method according to any one of embodiments 1-29, wherein the    high growth nutrient component is at a concentration of about 1.0    (v/v) to about 4.0% (v/v).-   31. The method according to any one of embodiments 1-29, wherein the    ammonium ferric citrate is at a concentration of about 0.1 mg/mL to    about 15 mg/mL.-   32. The method according to any one of embodiments 1-31, wherein the    enrichment media further comprises a growth enhancing agent.-   33. The method according to embodiment 32, wherein the growth    enhancing agent is a siderophore.-   34. The method according to embodiment 33, wherein the siderophore    is Aerobactin, Alcaligin, Azotobactin, Bacillibactin,    Desferrioxamine B, Desferrioxamine E, Enterobactin, Ferrichrome,    Ferrioxiamina-B, Ferrioxiamina-E, Fusarinine C, Mycobactin,    Ornibactin, Petrobactin, Pyoverdine, Pyochelin, Salmochelin,    Staphyloferring A, Vibriobactin, or Yersiniabactin.-   35. The method according to any one of embodiments 1-34, wherein    detection step (c) is performed using a sensor-based detection    method, a nucleic acid-based detection method, a protein-based    detection method, an activity-based detection method, or a    growth-based detection method.-   36. The method according to embodiment 35, wherein the sensor-based    detection method is an electrochemical detection method.-   37. The method according to embodiment 36, wherein the    electrochemical detection method comprises an enzymatic biosensor, a    DNA sensor, or an immunosensor.-   38. The method according to embodiment 36, wherein the    electrochemical detection method measures potentiometric parameters,    amperometric parameters, voltametric parameters,    impedimetric/conductimetric parameters, or any combination thereof.-   39. The method according to embodiment 35, wherein the nucleic    acid-based detection method comprises a DNA-based detection method    or an RNA-based detection method.-   40. The method according to embodiment 39, wherein the DNA-based    detection method comprises a Southern blot analysis, a PCR-based    assay, a sequence analysis, an immuno-based detection assay, or a    hybridization assays using FRET, polarization or other fluorescent,    chemiluminescent or bioiluminescent detection.-   41. The method according to embodiment 40, wherein the PCR-based    assay comprises a real-time PCR-based assay.-   42. The method according to embodiment 39, wherein the RNA-based    detection method comprises a Northern blot analysis, a RT-PCR-based    assay, a RNA sequence analysis, an immuno-based detection assay, or    a hybridization assays using FRET, polarization or other    fluorescent, chemiluminescent or bioiluminescent detection.-   43. The method according to embodiment 35, wherein the protein-based    detection method is a gel-based detection method, an immuno-based    detection method or a protein-interaction-based method.-   44. The method according to embodiment 43, wherein the immuno-based    detection method comprises a Western blot analysis, an ELISA, or an    immunoprecipitation assay.-   45. The method according to embodiment 35, wherein the    activity-based detection method comprises an enzymatic activity    assay or an assay based on protein function.-   46. The method according to embodiment 45, wherein the enzymatic    activity assay uses a spetrophotometric detection method measures a    disappearance of a substrate or a formation of a product.-   47. The method according to embodiment 35, wherein the growth-based    detection method comprises a plating assays measuring colony    formation or spectrophotometer assays measuring cell density.-   48. The method according to any one of embodiments 1-47, wherein the    pathogen is a prion, a virus, a bacterium, a fungus, a protazoan, a    helimenth, or a parasite.-   49. The method according to embodiment 48, wherein the virus belongs    to the family Adenoviridae, Picornaviridae, Herpesviridae,    Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae,    Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, or    Togaviridae.-   50. The method according to embodiment 48, wherein the bacteria    belongs to the genus Bacillus, Bordetella, Borrelia, Brucella,    Campylobacter, Chlamydia, Chlamydophila, Clostridium,    Corynebacterium, Enterobacter, Enterococcus, Escherichia,    Francisella, Haemophilus, Helicobacter, Legionella, Leptospira,    Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,    Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,    Treponema, Vibrio, or Yersinia.-   51. The method according to embodiment 48, wherein the fungus    belongs to the genus Asperfillus, Canidia, Cryptococcus,    Histoplasma, Pneumocystis, or Stachybotrys.-   52. The method according to embodiment 48, wherein the protozoan    belongs to the genus Acanthamoeba, Balamuthia, Cryptosporidium,    Dientamoeba, Endolimax, Entamoeba, Giardia, Iodamoeba, Leishmania,    Naegleria, Plasmodium, Sappinia, Toxoplasma, Trichomonas, or    Trypanosoma.-   53. A pathogen analysis kit comprising a pre-enrichment media and an    enrichment media.-   54. The pathogen analysis kit according to embodiment 53, further    comprising a detection solution.-   55. The pathogen analysis kit according to embodiment 53 or    embodiment 54, further and an electrochemical biosensor.-   56. The pathogen analysis kit according to any one of embodiments    53-55, further comprising immunmagnetic particles capable of binding    to a pathogen of interest.-   57. The pathogen analysis kit according to any one of embodiments    53-56, further comprising a magnetic source used to capture the    immunoparticles.-   58. The pathogen analysis kit according to any one of embodiments    53-57, further comprising an instrument which is capable of    measuring and/or analyzing potentiometric, voltammetric,    amperometric and/or impedance/conductance parameters.-   59. The pathogen analysis kit according to any one of embodiments    53-58, further comprising a suitable container.-   60. The pathogen analysis kit according to any one of embodiments    53-59, further comprising a label or an insert.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofthe disclosed subject matter. These examples should not be construed tolimit any of the embodiments described in the present specification,including those pertaining to the methods disclosed, the type ofdetection employed or the type of pathogen that can be detected usingthe methods disclosed herein.

Example 1 Preparation of Sensors and Electrochemical Readings

Enzyme-based electrochemical sensors were manufactured using a screenprinting process by depositing a series of layers of different materialon a substrate material. Initially, a layer of compound paste comprisingsilver is deposited directly onto a polyester substrate (MYLAR® A (500gauge); DuPont E. I. De Nemours & Co., Wilmington, Del.). A layer ofsilver/silver chloride, which functions as both the counter electrodeand reference electrode, is then deposited over the compound paste onone side to form a wall. The third layer comprised carbon graphite(Gwent Electronic Materials Ltd., Pontypool, UK) which functions as theworking electrode is deposited on the other side to form an oppossingwall. A channel is thus formed inbetween the silver/silver chloride walland the carbon graphite wall. A layer of insulating paste (GwentElectronic Materials Ltd., Pontypool, UK) is then deposited over thesilver/silver chloride and carbon graphite layers and apressure-sensitive adhesive (KIWO, Inc., Seabrook, Tex.) is thendeposited over this insulating paste layer. A sensor is then modified byadding a biological layer within the channel by incubating the sensor inan atmosphere saturated with 5% glutaraldehyde to 5% for 5 minutes atroom temperature in the presence of an enzyme solution comprisingglucose dehydrogenase (GDH), which is then dried for 30 minutes at 37°C. Finally, a layer of polyester (MELINEX®; DuPont E. I. De Nemours &Co., Wilmington, Del.) is placed on top of the insulating paste layer tocover the sensor and enclose the channel. The sensor is then heated at60° C. and pressure applied using a pressure roller to set the polyesterlayer. The sensor is stored in a dry place at 4° C. until needed.

Presence of the desired pathogen was detected by measuring theelectrochemical signal produced as a result of a redox reactionassociated with the conversion of glucose to gluconic acid. In thisdetection procedure, an aliquot of the enriched pathogen culture wasadded to a detection solution comprising p-aminophenyl phosphate (PAPH)and glucose. Alternatively, alfanaphtylphosphate may be used instead ofPAPH. A sensor as described above was inserted into this solution andelectrochemical signals were detected amperometrically under an appliedpotential of about 200 mV using a PalmSens3 (a potentiostat,galvanostat, and impedance analyzer) and its accompanying softwarePSTrace (PalmSens BV, Utrecht, Netherlands).

Briefly, the mechanism underlying the generation of an electrochemicalsignal is as follows. Pathogens detected using this procedure synthesizethe enzyme alkaline phosphatase (ALP), which is subsequently releasedinto the culture media. Addition of an enriched pathogen culturecomprising ALP results in the hydrolysis PAPH into PAP. GDH, immobilizedin the biological layer of the sensor, catalyzes the conversion ofglucose into gluconic acid, which is associated with a redox reactionthat oxidizes PAP into PIQ. The electrochemical signals detected by thesensor occurs when it measures the electrons generated when PIQ isreduced back to PAP.

Example 2 Relationship Between Measured Electrochemical Signal,Bacterial Population and Incubation Time

Generation of an electrochemical signal and its relationship with thepopulation size of bacteria was determined by measuring the currentgenerated by populations comprising different concentrations ofbacteria. A series of 50 μL detection solutions, each comprising 500 mMacetate buffer (pH 5.7), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mMglucose, was set up. Each detection solution was then mixed with a 1 mLsolution of phospho-buffered saline comprising bacteria (Salmonellatyphimurium) at one of the following concentrations: 1×10² cfu/mL, 1×10³cfu/mL, 1×10⁴ cfu/mL, 1×10⁵ cfu/mL, 1×10⁶ cfu/mL, 1×10⁷ cfu/mL, or 1×10⁸cfu/mL. The detection solution was then incubated at 37° C. for 30minutes. The electrochemical signal for each solution was detectedamperometrically under an applied potential of about 200 mV using aPalmSens3 as described above. As shown in FIG. 1, a bacterialconcentration of about 1×10⁴ cfu/mL produced a detectable signal ofabout 0.5 μA. In addition, a linear concentration-response curve wasobserved in a range of about 1×10⁵ cfu/mL to about 1×10⁷ cfu/mL. Theseresults indicate that and average bacterial concentration of about 1×10⁴cfu/mL can be detected using the electrochemical detection methoddisclosed herein.

Generation of an electrochemical signal and its relationship with theincubation time was determined by measuring the current generated by abacterial population incubated over time. A 225 mL culture mediacomprising buffered peptone and water was inoculated with 10 cfu/mL ofbacteria (S. typhimurium) and incubation at 37° C. over time. A 100 μLaliquot of this inoculated media was taken every two hours and added toa 50 μL detection solution comprising 500 mM acetate buffer (pH 5.7), 10mM magnesium chloride, 1.0 mM PAPH and 10 mM glucose. The detectionsolution was then incubated at 37° C. for 30 minutes. Theelectrochemical signal for each solution was detected amperometricallyunder an applied potential of about 200 mV using a PalmSens3 asdescribed above. As shown in FIG. 2, a detectable signal of about 0.5 μAwas detected after 4 hours of incubation. A linear time-response curvewas observed in a range of about 6 hours to about 8 hours. These resultsindicate that and average anodic current of >0.5 μA is indicative of thepresence of bacteria.

Example 3 Determination of the Presence of Salmonella in Pure Culture

Generation of an electrochemical signal by a bacteria from a pure samplewas determined by measuring the current generated by a defined bacterialpopulation of two different concentrations. A 225 mL pre-enrichmentculture media comprising buffered peptone and 0.4% (w/v) Tergitol-4 wasinoculated with either 20 cfu or 200 cfu of bacteria (S. typhimurium),mixed vigorously for 10 seconds, and incubation at 37° C. for about 14to about 17 hours.

After this pre-enrichment step, 1 mL of the pre-enrichment culture wastaken for each bacterial concentration and a 10 μL aliquot was added to990 μL of an enrichment culture media comprising 2.72% (w/v) ofRappaport-Vassiliadis Soya (pH 5.2) comprising 4.5 g/L soya peptone, 7.2g/L sodium chloride, 1.26 g/L potassium dihydrogen phosphate, 0.18 mg/Ldipotassium phosphate, 13.6 mg/L magnesium chloride and 0.036 g/Lmalachite green (Cultimed), supplemented with 0.62 mg/mL of ammoniumferric citrate (Sigma-Aldrich Corp., St. Louis, Mo.). The enrichmentculture for both concentrations were incubated for about 5 to about 7hours at 41.5° C. with circular stirring. After this enrichment step, 20μL of a solution 3.0×10⁸ anti-Salmonella immunomagnetic particles/mL(DYNABEADS® Anti-Salmonella, Life Technologies, Inc., Carlsbad, Calif.)was added to the enrichment cultures and incubated for 30 minutes atroom temperature with agitation circular. After this incubation time,the enrichment cultures were contacted with a magnet for 3 minutes inorder to localize the anti-Salmonella immunomagnetic particles withinthe container tube and the supernatant was extracted using 1 mLmicropipette. The supernatant was then added to 1 mL of 100 mM phosphatebuffer, mixed for 10 seconds, contacted with a magnet for 3 minutes andthe washed supernatant was extracted using 1 mL micropipette. Theprocessed supernatant was then tested to detect the presence of thebacteria using two different assays: 1) a plating assay; and 2) anelectrochemical detection assay disclosed herein.

To detect the presence of bacteria using a plating assay, a 100 μLaliquot of the processed supernatant was mixed into 500 μL of 100 mMbuffer phosphate (pH 7.4) and this mixture was plated on agar comprisinga selective chromogenic agent (Chromosalm MICROKIT®, LaboratoriosMICROKIT, S.L.). The agar plates were then incubated at 37° C. for about15 hours and identifying of colored bacterial colonies determined. Asshown in Table 1, growth of chromogenic bacterial colonies was observedfrom a supernatant sample derived from both the 20 cfu and 200 cfu ofpure inoculum cultures. These results indicate that the growth methodsdisclosed herein in conjunction with a plating assay can effectivelydetect the presence of a pathogen.

TABLE 1 Detection of Salmonella in pure inoculum sample Plating MethodElectrochemical method Pure Pure Pure Pure Ex- inoculum inoculuminoculum inoculum periment n 20 cfu 200 cfu 20 cfu 200 cfu 1 4 GrowthGrowth 2.37 ± 0.52 μA 2.45 ± 0.58 μA

To detect the presence of bacteria using a electrochemical detectionassay, a 100 μL aliquot of the processed supernatant was added to a 50μL detection solution comprising 500 mM acetate buffer (pH 5.7), 10 mMmagnesium chloride, 1.0 mM PAPH and 10 mM glucose. The detectionsolution was then incubated at 37° C. for 30 minutes. Theelectrochemical signal for each solution was detected amperometricallyunder an applied potential of about 200 mV verse Ag/AgCl for 30 secondsusing a PalmSens3 as described above. As shown in Table 1, a currentabove 2 μA was detected from a supernatant sample derived from both the20 cfu and 200 cfu of pure inoculum cultures. These results indicatethat the growth methods disclosed herein in conjunction with anelectrochemical detection assay disclosed herein can effectively detectthe presence of a pathogen.

Example 4 Determination of the Presence of Salmonella in Fecal Sample

Generation of an electrochemical signal by a bacteria from a samplematerial was determined by measuring the current generated from fecalmaterial. Twenty-five gram samples of fecal material, each suspected tocontain bacteria (S. typhimurium), were added into a 225 mLpre-enrichment culture media comprising buffered peptone and 0.4%Tergitol-4 and incubation at 37° C. for about 14 to about 15 hours.Fecal material samples proven not to contain bacteria (S. typhimurium)were also tested as a negative control. The procedures regardingpre-enrichment, enrichment, and immunoseparation for Experiment 1 inTable 2 were all performed as described above in Example 3. In addition,detection of the presence of the bacteria using a plating assay and anelectrochemical detection assay disclosed herein were performed asdescribed above in Example 3. For experiments 2 and 3 in Table 1, theimmunoseparation step was not performed. In these experiments, afterthis enrichment step, a 1 mL aliquot of enriched culture medium wascentrifuged and supernatant was extracted using 1 mL micropipette. Thesupernatant was then added to 1 mL of 100 mM phosphate buffer, mixed for10 seconds, centrifuged, and the washed supernatant was extracted using1 mL micropipette. The processed supernatant was then tested to detectthe presence of the bacteria using a plating assay and anelectrochemical detection assay disclosed herein as described above inExample 3.

As shown in Table 2, growth of chromogenic bacterial colonies wasobserved from a supernatant sample derived from fecal samplescontaminated with bacteria (S. typhimurium) while fecal samples notcontaminated with bacteria (S. typhimurium) did not exhibit anychromogenic bacterial colonies. In addition, currents above 2 μA weredetected from supernatant samples derived from fecal samplescontaminated with bacteria (S. typhimurium)(Table 2). However, as shownin Table 2, currents below 0.5 μA were measured from fecal samples notcontaminated with bacteria (S. typhimurium). These results indicate thata fecal sample contaminated with a pathogen can be identified anddistinguished from a fecal sample not contaminated with a pathogen usingthe methods disclosed herein.

TABLE 2 Detection of Salmonella in fecal sample Experi- Plating AssayElectrochemical Detection Assay ment n Fecal (+) Fecal (−) Fecal (+)Fecal (−) 1 4 Growth No Growth 1.97 ± 0.62 μA 0.30 ± 0.11 μA 2 4 GrowthNo Growth 2.36 ± 0.20 μA 0.25 ± 0.08 μA 3 4 Growth No Growth 3.33 ± 0.53μA 0.36 ± 0.08 μA

Example 5 Determination of the Presence of Salmonella in Chicken SkinSample

Generation of an electrochemical signal by a bacteria from a samplematerial was determined by measuring the current generated from skinsamples from chickens. Twenty-five gram samples of chicken skin, eachsuspected to contain bacteria (S. typhimurium), were added into a 225 mLpre-enrichment culture media comprising buffered peptone, 0.4% (v/v)brilliant green, 0.4% (w/v) Tergitol-4, mixed vigorously for 10 seconds,and then incubation at 37° C. for about 14 to about 15 hours. Chickenskin samples proven not to contain bacteria (S. typhimurium) were alsotested as a negative control. The procedures regarding pre-enrichment,enrichment, and immunoseparation for Experiment 1 in Table 2 were allperformed as described above in Example 3. In addition, detection of thepresence of the bacteria using a plating assay and an electrochemicaldetection assay disclosed herein were performed as described above inExample 3. For Experiment 2 in Table 1, the immunoseparation step wasnot performed. In this experiment, after this enrichment step, a 1 mLaliquot of enriched culture medium was centrifuged and supernatant wasextracted using 1 mL micropipette. The supernatant was then added to 1mL of 100 mM phosphate buffer, mixed for 10 seconds, centrifuged, andthe washed supernatant was extracted using 1 mL micropipette. Theprocessed supernatant was then tested to detect the presence of thebacteria using a plating assay and an electrochemical detection assaydisclosed herein as described above in Example 3.

As shown in Table 3, growth of chromogenic bacterial colonies wasobserved from a supernatant sample derived from chicken skin samplescontaminated with bacteria (S. typhimurium) while chicken skin samplesnot contaminated with bacteria (S. typhimurium) did not exhibit anychromogenic bacterial colonies. In addition, currents above 2 μA weredetected from supernatant samples derived from chicken skin samplescontaminated with bacteria (S. typhimurium)(Table 2). However, as shownin Table 3, currents below 0.5 μA were measured from chicken skinsamples not contaminated with bacteria (S. typhimurium). These resultsindicate that a chicken skin sample contaminated with a pathogen can beidentified and distinguished from a chicken skin sample not contaminatedwith a pathogen using the methods disclosed herein.

TABLE 3 Detection of Salmonella in chicken skin sample Ex- Plating AssayElectrochemical Detection Assay periment n Skin (+) Skin (−) Skin (+)Skin (−) 1 4 Growth No Growth 1.84 ± 0.61 μA 0.24 ± 0.10 μA 2 4 GrowthNo Growth 2.35 ± 0.49 μA 0.35 ± 0.22 μA

Example 6 Determination of the Presence of Salmonella in FoodstuffSample

Generation of an electrochemical signal by a bacteria from a samplematerial was determined by measuring the current generated fromfoodstuff samples of corn. Twenty-five gram samples of chicken skin,each suspected to contain bacteria (S. typhimurium), were added into a225 mL pre-enrichment culture media comprising buffered peptone, 0.4%(v/v) brilliant green, 0.4% (w/v) Tergitol-4, mixed vigorously for 10seconds, and then incubation at 37° C. for about 14 to about 15 hours.Foodstuff samples proven not to contain bacteria (S. typhimurium) werealso tested as a negative control. The procedures regardingpre-enrichment and enrichment were all performed as described above inExample 3, however, the immunoseparation step was not performed. In thisexperiment, after this enrichment step, a 1 mL aliquot of enrichedculture medium was centrifuged and supernatant was extracted using 1 mLmicropipette. The supernatant was then added to 1 mL of 100 mM phosphatebuffer, mixed for 10 seconds, centrifuged, and the washed supernatantwas extracted using 1 mL micropipette. The processed supernatant wasthen tested to detect the presence of the bacteria using a plating assayand an electrochemical detection assay disclosed herein as describedabove in Example 3.

As shown in Table 4, growth of chromogenic bacterial colonies wasobserved from a supernatant sample derived from foodstuff samplescontaminated with bacteria (S. typhimurium) while foodstuff samples notcontaminated with bacteria (S. typhimurium) did not exhibit anychromogenic bacterial colonies. In addition, currents above 2 μA weredetected from supernatant samples derived from foodstuff samplescontaminated with bacteria (S. typhimurium)(Table 2). However, as shownin Table 4, currents below 0.5 μA were measured from foodstuff samplesnot contaminated with bacteria (S. typhimurium). These results indicatethat a foodstuff sample contaminated with a pathogen can be identifiedand distinguished from a foodstuff sample not contaminated with apathogen using the methods disclosed herein.

TABLE 4 Detection of Salmonella in foodstuff sample Ex- peri- PlatingAssay Electrochemical Detection Assay ment n Foodstuff (+) Foodstuff (−)Foodstuff (+) Foodstuff (−) 1 4 Growth No Growth 2.38 ± 0.62 μA 0.62 ±0.06 μA

Example 7 Determination of the Presence of Salmonella UsingSpectrophotometer Assay

Generation of an electrochemical signal by a bacteria from a samplematerial was determined by measuring enzymatic activity using aspectrophotometer. Twenty-five gram samples of fecal material, eachsuspected to contain bacteria (S. typhimurium), were added into a 225 mLpre-enrichment culture media comprising buffered peptone and 0.4%Tergitol-4 and incubation at 37° C. for about 16 hours. Fecal materialsamples proven not to contain bacteria (S. typhimurium) were also testedas a negative control. The procedures regarding enrichment andimmunopurification were performed as described above in Example 3,except that an aliquoit of culture media was removed from the enrichmentculture at each hour for the first eight hours post-inoculation and thenagain at 24 hours post-inoculation. The immunomagnetic particles wereresuspended in 50 μL of 200 mM phosphate buffer (pH 5.7) and incubatedat room temperature overnight. The incubated sample was then contactedwith a magnet for 3 minutes in order to localize the immunomagneticparticles within the container tube and the supernatant was extractedusing 1 mL micropipette. The 50 μL aliquot of supernatant was then addedto a well from a 96-well plate containing 50 μL of substrate solutioncomprising 5.4 mM p-Nitrophenol phosphate. A standard solution was alsoprepared and comprised 0.05 mM p-Nitrophenol in 0.5 N sodium hydroxide.Control wells were also established. Negative control wells included 50μL of 200 mM phosphate buffer (pH 5.7) and 50 μL of substrate solution.Positive control wells included 2 μL of 0.5-3 units/mg of acidphosphatase, 48 μL of 200 mM phosphate buffer (pH 5.7) and 50 μL ofsubstrate solution. The plates were incubated at 37° C. for 30 minutesand then a 200 μL stop solution of 0.5 N sodium hydroxide. In thepresence of alkaline phosphatase, the p-Nitrophenol phosphate will beconverted into p-Nitrophenol, resulting in a color change. Theabsorption at 405 mn was measured for each well using aspectrophotometer. The amount of alkaline phosphatase activity for eachaliquot was determined based on the standard curve generated from thestandard solution.

As shown in FIG. 3, a linear time-response curve was observed for thecontaminated fecal samples for the first 6 hours with the signal thenplateauing for the remainder of the time course. The blank and negativecontrol showed no enzyme activity during the entire time course. Theseresults indicate that the presence of bacteria can be detecting using aspectrophotometric measurements.

Example 8 Determination of the Presence of Salmonella Using PCR Assay

Generation of an electrochemical signal by a bacteria from a samplematerial was determined by measuring enzymatic activity using aspectrophotometer. Twenty-five gram samples of fecal material, eachsuspected to contain bacteria (S. typhimurium), were added into a 225 mLpre-enrichment culture media comprising buffered peptone and 0.4%Tergitol-4 and incubation at 37° C. for about 16 hours. Fecal materialsamples proven not to contain bacteria (S. typhimurium) were also testedas a negative control. The procedures regarding enrichment andimmunopurification were performed as described above in Example 3. Theimmunomagnetic particles were resuspended in 200 μL of DNAase-free waterand then heated for 30 minutes at 98° C. to lyse the cells. Aftercentrifugation at 6000 rpm for 10 minutes at 4° C., the supernatant wastransferred to a new tube. A 5 μL aliquot was added to a 15 μL PCRreaction mixture containing 10 μM of a forward and reverse primerspecific for and diagnostic of S. typhimurium. PCR was performed asfollows: 1 cycle at 95° C. for 10 minutes and up to 40 cycles of 95° C.for 30 seconds and 60° C. for 1 minute. The level of target DNAamplified during each cycle was measured by detecting fluorescence andrecording the RFU. Alternatively, the assay was performed without animmunopurification step. In this case, after the incubation period forthe enrichment step was completed, the culture was centrifugation at6000 rpm for 10 minutes at 4° C., the supernatant was transferred to anew tube, and a 5 μL aliquot used for the PCR reaction.

As shown in FIG. 4A-C, detection of target DNA was initially observedafter 20 cycles of amplification. Amplification of target DNA exhibiteda sigmoidal curve that typically began plateauing by cycle 30. Thenegative control showed no detectable level of target DNA amplificationduring the entire time course. These results indicate that the presenceof bacteria can be detecting using a PCR-based assay.

Example 9 Determination of the Presence of Other Organisms

Generation of an electrochemical signal by different bacteria wasdetermined using three different detection solutions at variousbacterial concentrations. A series of 50 μL detection solutions wasestablished for three different pH values as follows: 1) 200 mM acetatebuffer (pH 5.7), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mMglucose; 2) 200 mM phosphate buffer (pH 7.0), 10 mM magnesium chloride,1.0 mM PAPH and 10 mM glucose; or 3) 200 mM TRIS buffer (pH 9.8), 10 mMmagnesium chloride, 1.0 mM PAPH and 10 mM glucose. Each detectionsolution was then mixed with a 1 mL solution of phospho-buffered salinecomprising a bacteria at one of the following concentrations: 1×10²cfu/mL, 1×10³ cfu/mL, 1×10⁴ cfu/mL, 1×10⁵ cfu/mL, 1×10⁶ cfu/mL, 1×10⁷cfu/mL, or 1×10⁸ cfu/mL. The bacteria tested were as follows:Campylobacter jejuni Cluster 1, Campylobacter jejuni ST45, Campylobactercoli, Escherichia coli, Salmonella typhimurium, Pseudomonas sp.,Pseudomonas aureginosa, Enterobacter cloacae, MRSA, Proteus sp.,Bacillus thuringiensis, Citrobacter sp., Shigella sp., Staphylococccusaureus, Klebsiella pneumoniae, and Enterococcus faecalis. After thebacteria was added, these detection solution was mixed for 10 secondsand then incubated at 37° C. for 30 minutes. Detection of the presenceof the bacteria using an electrochemical detection assay disclosedherein were performed as described above in Example 3.

As shown in FIGS. 5-7, a bacterial concentration of about 1×10⁴ cfu/mLproduced a detectable signal of about 0.5 μA for all bacteria tested,although pH of the detection solution had an influence on thesensitivity of detection for certain bacterial species. In addition, alinear concentration-response curve was observed in a range of about1×10⁵ cfu/mL to about 1×10⁷ cfu/mL. These results indicate that andaverage bacterial concentration of about 1×10⁴ cfu/mL can be detectedusing the electrochemical detection method disclosed herein.

TABLE 5 Electrochemical detection of other pathogens Organism IntensityAssay pH 5.7 pH 7.0 pH 9.8 C. jejuni Cluster 1 >1 μA cfu/mL — — 2.11 ×10⁵ μA — — 1.46 ± 0.35 <1 μA cfu/mL 4.94 × 10⁶ 1.00 × 10⁷ 2.11 × 10⁴ μA0.27 ± 0.05 0.70 ± 0.08 0.89 ± 0.07 C. jejuni ST45 >1 μA cfu/mL — — 2.34× 10⁵ μA — — 1.54 ± 0.17 <1 μA cfu/mL 8.33 × 10⁵ 2.34 × 10⁶ 2.34 × 10⁴μA 0.29 ± 0.06 0.18 ± 0.04 0.53 ± 0.07 C. coli >1 μA cfu/mL — — 1.53 ×10⁵ μA — — 1.52 ± 0.28 <1 μA cfu/mL 3.00 × 10⁴ 7.17 × 10⁵ 1.53 × 10⁴ μA0.24 ± 0.07 0.19 ± 0.04 0.62 ± 0.07 E. coli >1 μA cfu/mL 5.40 × 10⁷ 2.04× 10⁸ — μA 3.25 ± 0.36 1.44 ± 0.45 — <1 μA cfu/mL 5.40 × 10⁶ 2.41 × 10⁶4.00 × 10⁷ μA 0.74 ± 0.10 0.28 ± 0.00 0.73 ± 0.12 S. typhimurium >1 μAcfu/mL 1.72 × 10⁶ 1.65 × 10⁶ 3.39 × 10⁷ μA 1.60 ± 0.26 1.26 ± 0.14 2.29± 0.42 <1 μA cfu/mL 1.72 × 10⁵ 1.65 × 10⁵ 3.39 × 10⁶ μA 0.41 ± 0.05 0.25± 0.01 0.47 ± 0.04 Pseudomonas sp. >1 μA cfu/mL 1.63 × 10⁶ 2.45 × 10⁶3.43 × 10⁶ μA 2.46 ± 0.35 1.45 ± 0.15 1.85 ± 0.46 <1 μA cfu/mL 1.63 ×10⁵ 2.45 × 10⁵ 3.43 × 10⁵ μA 0.59 ± 0.03 0.47 ± 0.11 0.87 ± 0.05 P.aureginosa >1 μA cfu/mL * — — μA * — — <1 μA cfu/mL 2.18 × 10⁶ 1.65 ×10⁶ 1.50 × 10⁶ μA 0.93 ± 0.01 0.74 ± 0.01 0.77 ± 0.05 E. cloacae >1 μAcfu/mL 8.99 × 10⁷ — — μA 1.37 ± 0.11 — — <1 μA cfu/mL 8.99 × 10⁶ 9.88 ×10⁷ 1.28 × 10⁸ μA 0.27 ± 0.02 0.25 ± 0.02 0.41 ± 0.05 MRSA >1 μA cfu/mL1.01 × 10⁶ 3.64 × 10⁶ — μA 1.33 ± 0.29 1.19 ± 0.10 — <1 μA cfu/mL 1.01 ×10⁵ 3.64 × 10⁵ 3.23 × 10⁷ μA 0.32 ± 0.04 0.33 ± 0.04 0.75 ± 0.06 Proteussp. >1 μA cfu/mL 1.70 × 10⁸ 4.92 × 10⁷ 5.79 × 10⁷ μA 4.10 ± 0.31 2.82 ±0.16 1.36 ± 0.05 <1 μA cfu/mL 1.70 × 10⁷ 4.92 × 10⁶ 5.79 × 10⁵ μA 0.86 ±0.09 0.41 ± 0.01 0.85 ± 0.05 B. thuringiensis >1 μA cfu/mL 2.11 × 10⁵3.11 × 10⁶ — μA 1.22 ± 0.24 2.08 ± 0.10 — <1 μA cfu/mL 2.11 × 10⁴ 3.11 ×10⁵ 1.93 × 10⁶ μA 0.26 ± 0.04 0.55 ± 0.07 0.74 ± 0.06 Citrobacter sp. >1μA cfu/mL 4.23 × 10⁷ 2.06 × 10⁸ — μA 1.27 ± 0.08 2.40 ± 0.08 — <1 μAcfu/mL 4.23 × 10⁶ 2.06 × 10⁷ 7.62 × 10⁷ μA 0.27 ± 0.04 0.41 ± 0.05 0.62± 0.11 Shigella sp. >1 μA cfu/mL 8.66 × 10⁷ 9.87 × 10⁷ — μA 4.46 ± 0.441.68 ± 0.31 — <1 μA cfu/mL 8.66 × 10⁶ 9.87 × 10⁶ 1.60 × 10⁸ μA 0.78 ±0.09 0.35 ± 0.05 0.69 ± 0.15 S. aureus >1 μA cfu/mL 2.63 × 10⁷ 2.15 ×10⁷ — μA 2.99 ± 0.23 1.22 ± 0.20 — <1 μA cfu/mL 2.63 × 10⁶ 2.15 × 10⁶9.44 × 10⁶ μA 0.56 ± 0.05 0.31 ± 0.07 0.49 ± 0.13 K. pneumoniae >1 μAcfu/mL 6.20 × 10⁶ 1.12 × 10⁸ — μA 1.74 ± 0.08 2.56 ± 0.32 — <1 μA cfu/mL6.20 × 10⁵ 1.12 × 10⁷ 4.43 × 10⁷ μA 0.41 ± 0.02 0.49 ± 0.13 0.82 ± 0.17E. faecalis >1 μA cfu/mL 4.84 × 10⁷ — — μA 3.82 ± 0.92 — — <1 μA cfu/mL4.84 × 10⁶ 7.33 × 10⁷ 4.72 × 10⁷ μA 0.92 ± 0.01 0.54 ± 0.03 0.86 ± 0.08

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

1. A method of detecting Enterobacter sakazakii in a food sample, themethod comprising the steps of: a) incubating the sample in a mediacomprising a peptone nutrient component and a cell wall growthinhibiting agent, wherein the incubation is at about 37° C. to about 45°C. for about 5 hours; b) incubating an aliquot of the media in aglucose-based reaction solution, wherein the incubation is at about 37°C. to about 45° C. for about 16 hours; and c) detecting the presence ofabsence of Enterobacter sakazakii by analyzing an aliquot of thereaction media from step (b) in an electrochemical sensor.